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Experiment: Microarray analysis of HopAO1 transgenic Arabidopsis plants

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-416

This experiment was performed to identify gene expression changes resulting from the expression of the Pseudomonas syringae pv. tomato (Pst) strain DC3000 type III effector tyrosine phosphatase HopAO1 in stable Arabidopsis transgenic lines. Wild-type (Col gl1) plants and transgenic plants expressing HopAO1 or a mutant derivative, HopAO1 C378S, lacking phosphatase catalytic activity were vacuum-infiltrated with either a mock inoculum of sterile water or a suspension of the Pst DC3000 hrpA mutant and changes in gene expression in infiltrated leaf tissues were investigated 7 hours after inoculation using Affymetrix ATH1 genechips.

About the Experimenter

Name:Dr. William Underwood
Head of Lab Name:Dr. Sheng Yang He
Lab: DOE Plant Research Laboratory
Institute: Michigan State University
Address:DOE Plant Research Laboratory
Michigan State University
East Lansing, MI
Postcode: 48824
Country: USA
 
Telephone Number: 5173539182

All of the data available in this website/database is free, and you are free to do whatever you please with it. If you intend to publish work based on any of this data, please acknowledge us, contact the experimenter above, and either acknowledge them or use them as co-authors in the work.

About this Experiment

Experiment Type: genetic_modification_design; pathogenicity_design
Number of Slides:18
 
Experimental Parameters:
parametergenetic_modification
parameterinfect
Quality Control Measures Taken:
References:
 
Other Information:

Slides in this Experiment

Hybridisation Set: Underwood: Microarray analysis of HopAO1 transgenic Arabidopsis plants_genome

Slide: Underwood_2-1_Col-gl1-mock-inocu_Rep1_ATH1

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Underwood_2-1_Col-gl1-mock-inocu_Rep1_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Underwood_2-1_Col-gl1-mock-inocu_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-5
Stock Code: N1644
Growth Conditions:
stratificationStratified 3 days at 4degC
Growth protocolArabidopsis thaliana accession Col-gl1 (Col-5) plants were grown in soil in growth chambers with a day/night cycle of 12 h/12 h, a light intensity of 100 µE, and a constant temperature of 20°C. Four-to 5-week-old plants were used for experiments.
LocationGrowth chamber
Growth substratecommercial soil
Average humidity80%
Average temperature20oC
Developmental Stage:
Growth Stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.70
Tissue: Leaves
in vivo Treatment:
treatmentPlants were inoculated by vacuum-infiltration with a mock inoculum of sterile water containing the surfactant Silwet L-77 at a concentration of .004%. Inoculated leaf tissue from at least 15 plants was collected for RNA isolation.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.529162
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.03
NoiseAvg:3.44,Stdev:0.08,Max:3.6,Min:3.2
BackgroundAvg:104.33,Stdev:0.64,Max:106.0,Min:103.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.529162
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Underwood_2-2_Col-gl1-mock-inocu_Rep2_ATH1

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Underwood_2-2_Col-gl1-mock-inocu_Rep2_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Underwood_2-2_Col-gl1-mock-inocu_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-5
Stock Code: N1644
Growth Conditions:
stratificationStratified 3 days at 4degC
Growth protocolArabidopsis thaliana accession Col-gl1 (Col-5) plants were grown in soil in growth chambers with a day/night cycle of 12 h/12 h, a light intensity of 100 µE, and a constant temperature of 20°C. Four-to 5-week-old plants were used for experiments.
LocationGrowth chamber
Growth substratecommercial soil
Average humidity80%
Average temperature20oC
Developmental Stage:
Growth Stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.70
Tissue: Leaves
in vivo Treatment:
treatmentPlants were inoculated by vacuum-infiltration with a mock inoculum of sterile water containing the surfactant Silwet L-77 at a concentration of .004%. Inoculated leaf tissue from at least 15 plants was collected for RNA isolation.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.641688
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.06
NoiseAvg:3.34,Stdev:0.13,Max:3.7,Min:3.0
BackgroundAvg:100.47,Stdev:1.35,Max:103.5,Min:96.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.641688
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Underwood_2-3_Col-gl1-mock-inocu_Rep3_ATH1

Add to Slide Selection

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Underwood_2-3_Col-gl1-mock-inocu_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Underwood_2-3_Col-gl1-mock-inocu_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-5
Stock Code: N1644
Growth Conditions:
stratificationStratified 3 days at 4degC
Growth protocolArabidopsis thaliana accession Col-gl1 (Col-5) plants were grown in soil in growth chambers with a day/night cycle of 12 h/12 h, a light intensity of 100 µE, and a constant temperature of 20°C. Four-to 5-week-old plants were used for experiments.
LocationGrowth chamber
Growth substratecommercial soil
Average humidity80%
Average temperature20oC
Developmental Stage:
Growth Stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.70
Tissue: Leaves
in vivo Treatment:
treatmentPlants were inoculated by vacuum-infiltration with a mock inoculum of sterile water containing the surfactant Silwet L-77 at a concentration of .004%. Inoculated leaf tissue from at least 15 plants was collected for RNA isolation.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.591805
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.64
NoiseAvg:2.95,Stdev:0.17,Max:3.8,Min:2.8
BackgroundAvg:89.62,Stdev:2.34,Max:98.3,Min:85.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.591805
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Underwood_2-4_HopAO1-mock-inocu_Rep1_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Underwood_2-4_HopAO1-mock-inocu_Rep1_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Underwood_2-4_HopAO1-mock-inocu_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: HopAO1
Stock Code:
Genetic Background: Col-5
Growth Conditions:
stratificationStratified 3 days at 4degC
Growth protocolArabidopsis thaliana accession Col-gl1 (Col-5) plants were grown in soil in growth chambers with a day/night cycle of 12 h/12 h, a light intensity of 100 µE, and a constant temperature of 20°C. Four-to 5-week-old plants were used for experiments.
LocationGrowth chamber
Growth substratecommercial soil
Average humidity80%
Average temperature20oC
Developmental Stage:
Growth Stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.70
Genetic Variation: T-DNA gene knockout
Tissue: Leaves
in vivo Treatment:
treatmentPlants were inoculated by vacuum-infiltration with a mock inoculum of sterile water containing the surfactant Silwet L-77 at a concentration of .004%. Inoculated leaf tissue from at least 15 plants was collected for RNA isolation
Additional Organism Information:
allele knocked outHopAO1 from Pst DC3000
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.625148
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.12
NoiseAvg:3.04,Stdev:0.26,Max:4.6,Min:2.8
BackgroundAvg:97.57,Stdev:0.54,Max:99.5,Min:95.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.625148
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Underwood_2-5_HopAO1-mock-inocu_Rep2_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Underwood_2-5_HopAO1-mock-inocu_Rep2_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Underwood_2-5_HopAO1-mock-inocu_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: HopAO1
Stock Code:
Genetic Background: Col-5
Growth Conditions:
stratificationStratified 3 days at 4degC
Growth protocolArabidopsis thaliana accession Col-gl1 (Col-5) plants were grown in soil in growth chambers with a day/night cycle of 12 h/12 h, a light intensity of 100 µE, and a constant temperature of 20°C. Four-to 5-week-old plants were used for experiments.
LocationGrowth chamber
Growth substratecommercial soil
Average humidity80%
Average temperature20oC
Developmental Stage:
Growth Stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.70
Genetic Variation: T-DNA gene knockout
Tissue: Leaves
in vivo Treatment:
treatmentPlants were inoculated by vacuum-infiltration with a mock inoculum of sterile water containing the surfactant Silwet L-77 at a concentration of .004%. Inoculated leaf tissue from at least 15 plants was collected for RNA isolation
Additional Organism Information:
allele knocked outHopAO1 from Pst DC3000
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.715875
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.59
NoiseAvg:3.71,Stdev:0.15,Max:4.2,Min:3.4
BackgroundAvg:114.58,Stdev:1.01,Max:117.6,Min:112.7
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.715875
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Underwood_2-6_HopAO1-mock-inocu_Rep3_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Underwood_2-6_HopAO1-mock-inocu_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Underwood_2-6_HopAO1-mock-inocu_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: HopAO1
Stock Code:
Genetic Background: Col-5
Growth Conditions:
stratificationStratified 3 days at 4degC
Growth protocolArabidopsis thaliana accession Col-gl1 (Col-5) plants were grown in soil in growth chambers with a day/night cycle of 12 h/12 h, a light intensity of 100 µE, and a constant temperature of 20°C. Four-to 5-week-old plants were used for experiments.
LocationGrowth chamber
Growth substratecommercial soil
Average humidity80%
Average temperature20oC
Developmental Stage:
Growth Stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.70
Genetic Variation: T-DNA gene knockout
Tissue: Leaves
in vivo Treatment:
treatmentPlants were inoculated by vacuum-infiltration with a mock inoculum of sterile water containing the surfactant Silwet L-77 at a concentration of .004%. Inoculated leaf tissue from at least 15 plants was collected for RNA isolation
Additional Organism Information:
allele knocked outHopAO1 from Pst DC3000
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.86118
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ1.96
NoiseAvg:1.87,Stdev:0.07,Max:2.1,Min:1.7
BackgroundAvg:75.09,Stdev:0.59,Max:76.7,Min:73.8
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.861180
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Underwood_2-7_HopAO1-C378S-mock-inocu_Rep1_ATH1

Add to Slide Selection

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Underwood_2-7_HopAO1-C378S-mock-inocu_Rep1_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Underwood_2-7_HopAO1-C378S-mock-inocu_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: HopAO1
Stock Code:
Genetic Background: Col-5
Growth Conditions:
stratificationStratified 3 days at 4degC
Growth protocolArabidopsis thaliana accession Col-gl1 (Col-5) plants were grown in soil in growth chambers with a day/night cycle of 12 h/12 h, a light intensity of 100 µE, and a constant temperature of 20°C. Four-to 5-week-old plants were used for experiments.
LocationGrowth chamber
Growth substratecommercial soil
Average humidity80%
Average temperature20oC
Developmental Stage:
Growth Stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.70
Genetic Variation: T-DNA gene knockout
Tissue: Leaves
in vivo Treatment:
treatmentPlants were inoculated by vacuum-infiltration with a mock inoculum of sterile water containing the surfactant Silwet L-77 at a concentration of .004%. Inoculated leaf tissue from at least 15 plants was collected for RNA isolation.
Additional Organism Information:
allele knocked outHopAO1 C378S from Pst DC3000
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.548012
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.84
NoiseAvg:2.95,Stdev:0.08,Max:3.2,Min:2.7
BackgroundAvg:99.74,Stdev:0.65,Max:101.2,Min:97.8
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.548012
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Underwood_2-8_HopAO1-C378S-mock-inocu_Rep2_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Underwood_2-8_HopAO1-C378S-mock-inocu_Rep2_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Underwood_2-8_HopAO1-C378S-mock-inocu_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: HopAO1
Stock Code:
Genetic Background: Col-5
Growth Conditions:
stratificationStratified 3 days at 4degC
Growth protocolArabidopsis thaliana accession Col-gl1 (Col-5) plants were grown in soil in growth chambers with a day/night cycle of 12 h/12 h, a light intensity of 100 µE, and a constant temperature of 20°C. Four-to 5-week-old plants were used for experiments.
LocationGrowth chamber
Growth substratecommercial soil
Average humidity80%
Average temperature20oC
Developmental Stage:
Growth Stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.70
Genetic Variation: T-DNA gene knockout
Tissue: Leaves
in vivo Treatment:
treatmentPlants were inoculated by vacuum-infiltration with a mock inoculum of sterile water containing the surfactant Silwet L-77 at a concentration of .004%. Inoculated leaf tissue from at least 15 plants was collected for RNA isolation.
Additional Organism Information:
allele knocked outHopAO1 C378S from Pst DC3000
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.647491
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.94
NoiseAvg:2.72,Stdev:0.15,Max:3.2,Min:2.4
BackgroundAvg:92.55,Stdev:0.66,Max:93.7,Min:90.6
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.647491
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Underwood_2-9_HopAO1-C378S-mock-inocu_Rep3_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Underwood_2-9_HopAO1-C378S-mock-inocu_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Underwood_2-9_HopAO1-C378S-mock-inocu_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: HopAO1
Stock Code:
Genetic Background: Col-5
Growth Conditions:
stratificationStratified 3 days at 4degC
Growth protocolArabidopsis thaliana accession Col-gl1 (Col-5) plants were grown in soil in growth chambers with a day/night cycle of 12 h/12 h, a light intensity of 100 µE, and a constant temperature of 20°C. Four-to 5-week-old plants were used for experiments.
LocationGrowth chamber
Growth substratecommercial soil
Average humidity80%
Average temperature20oC
Developmental Stage:
Growth Stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.70
Genetic Variation: T-DNA gene knockout
Tissue: Leaves
in vivo Treatment:
treatmentPlants were inoculated by vacuum-infiltration with a mock inoculum of sterile water containing the surfactant Silwet L-77 at a concentration of .004%. Inoculated leaf tissue from at least 15 plants was collected for RNA isolation.
Additional Organism Information:
allele knocked outHopAO1 C378S from Pst DC3000
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.733118
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.32
NoiseAvg:2.20,Stdev:0.07,Max:2.4,Min:2.0
BackgroundAvg:82.21,Stdev:0.51,Max:83.2,Min:80.8
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.733118
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Underwood_2-10_Col-gl1-hrpA-inocu_Rep1_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Underwood_2-10_Col-gl1-hrpA-inocu_Rep1_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Underwood_2-10_Col-gl1-hrpA-inocu_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-5
Stock Code: N1644
Growth Conditions:
stratificationStratified 3 days at 4degC
Growth protocolArabidopsis thaliana accession Col-gl1 (Col-5) plants were grown in soil in growth chambers with a day/night cycle of 12 h/12 h, a light intensity of 100 µE, and a constant temperature of 20°C. Four-to 5-week-old plants were used for experiments.
LocationGrowth chamber
Growth substratecommercial soil
Average humidity80%
Average temperature20oC
Developmental Stage:
Growth Stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.70
Tissue: Leaves
in vivo Treatment:
treatmentPlants were inoculated by vacuum-infiltration with a 1x10e8 cfu/ml suspension of the Pst DC3000 hrpA mutant in sterile water containing .004% Silwet L-77. Leaf tissue from at least 15 plants was collected 7 hours after inoculation for RNA isolation.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.46758
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.32
NoiseAvg:4.10,Stdev:0.34,Max:5.7,Min:3.6
BackgroundAvg:116.09,Stdev:1.26,Max:119.8,Min:112.5
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.467580
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Underwood_2-11_Col-gl1-hrpA-inocu_Rep2_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Underwood_2-11_Col-gl1-hrpA-inocu_Rep2_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Underwood_2-11_Col-gl1-hrpA-inocu_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-5
Stock Code: N1644
Growth Conditions:
stratificationStratified 3 days at 4degC
Growth protocolArabidopsis thaliana accession Col-gl1 (Col-5) plants were grown in soil in growth chambers with a day/night cycle of 12 h/12 h, a light intensity of 100 µE, and a constant temperature of 20°C. Four-to 5-week-old plants were used for experiments.
LocationGrowth chamber
Growth substratecommercial soil
Average humidity80%
Average temperature20oC
Developmental Stage:
Growth Stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.70
Tissue: Leaves
in vivo Treatment:
treatmentPlants were inoculated by vacuum-infiltration with a 1x10e8 cfu/ml suspension of the Pst DC3000 hrpA mutant in sterile water containing .004% Silwet L-77. Leaf tissue from at least 15 plants was collected 7 hours after inoculation for RNA isolation.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.949328
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.36
NoiseAvg:2.45,Stdev:0.13,Max:3.2,Min:2.1
BackgroundAvg:84.07,Stdev:1.30,Max:87.9,Min:81.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.949328
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Underwood_2-12_Col-gl1-hrpA-inocu_Rep3_ATH1

Add to Slide Selection

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Underwood_2-12_Col-gl1-hrpA-inocu_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Underwood_2-12_Col-gl1-hrpA-inocu_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-5
Stock Code: N1644
Growth Conditions:
stratificationStratified 3 days at 4degC
Growth protocolArabidopsis thaliana accession Col-gl1 (Col-5) plants were grown in soil in growth chambers with a day/night cycle of 12 h/12 h, a light intensity of 100 µE, and a constant temperature of 20°C. Four-to 5-week-old plants were used for experiments.
LocationGrowth chamber
Growth substratecommercial soil
Average humidity80%
Average temperature20oC
Developmental Stage:
Growth Stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.70
Tissue: Leaves
in vivo Treatment:
treatmentPlants were inoculated by vacuum-infiltration with a 1x10e8 cfu/ml suspension of the Pst DC3000 hrpA mutant in sterile water containing .004% Silwet L-77. Leaf tissue from at least 15 plants was collected 7 hours after inoculation for RNA isolation.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.541427
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.25
NoiseAvg:2.38,Stdev:0.07,Max:2.6,Min:2.2
BackgroundAvg:81.75,Stdev:0.38,Max:82.6,Min:80.7
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.541427
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Underwood_2-13_HopAO1-hrpA-inocu_Rep1_ATH1

Add to Slide Selection

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Underwood_2-13_HopAO1-hrpA-inocu_Rep1_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Underwood_2-13_HopAO1-hrpA-inocu_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: HopAO1
Stock Code:
Genetic Background: Col-5
Growth Conditions:
stratificationStratified 3 days at 4degC
Growth protocolArabidopsis thaliana accession Col-gl1 (Col-5) plants were grown in soil in growth chambers with a day/night cycle of 12 h/12 h, a light intensity of 100 µE, and a constant temperature of 20°C. Four-to 5-week-old plants were used for experiments.
LocationGrowth chamber
Growth substratecommercial soil
Average humidity80%
Average temperature20oC
Developmental Stage:
Growth Stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.70
Genetic Variation: T-DNA gene knockout
Tissue: Leaves
in vivo Treatment:
treatmentPlants were inoculated by vacuum-infiltration with a 1x10e8 cfu/ml suspension of the Pst DC3000 hrpA mutant in sterile water containing .004% Silwet L-77. Leaf tissue from at least 15 plants was collected 7 hours after inoculation for RNA isolation.
Additional Organism Information:
allele knocked outHopAO1 from Pst DC3000
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.530118
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.22
NoiseAvg:3.15,Stdev:0.09,Max:3.4,Min:2.8
BackgroundAvg:105.34,Stdev:0.56,Max:107.0,Min:103.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.530118
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Underwood_2-14_HopAO1-hrpA-inocu_Rep2_ATH1

Add to Slide Selection

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Underwood_2-14_HopAO1-hrpA-inocu_Rep2_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Underwood_2-14_HopAO1-hrpA-inocu_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: HopAO1
Stock Code:
Genetic Background: Col-5
Growth Conditions:
stratificationStratified 3 days at 4degC
Growth protocolArabidopsis thaliana accession Col-gl1 (Col-5) plants were grown in soil in growth chambers with a day/night cycle of 12 h/12 h, a light intensity of 100 µE, and a constant temperature of 20°C. Four-to 5-week-old plants were used for experiments.
LocationGrowth chamber
Growth substratecommercial soil
Average humidity80%
Average temperature20oC
Developmental Stage:
Growth Stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.70
Genetic Variation: T-DNA gene knockout
Tissue: Leaves
in vivo Treatment:
treatmentPlants were inoculated by vacuum-infiltration with a 1x10e8 cfu/ml suspension of the Pst DC3000 hrpA mutant in sterile water containing .004% Silwet L-77. Leaf tissue from at least 15 plants was collected 7 hours after inoculation for RNA isolation.
Additional Organism Information:
allele knocked outHopAO1 from Pst DC3000
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.706818
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.54
NoiseAvg:2.76,Stdev:0.06,Max:2.9,Min:2.6
BackgroundAvg:90.68,Stdev:0.61,Max:92.3,Min:89.4
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.706818
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Underwood_2-15_HopAO1-hrpA-inocu_Rep3_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Underwood_2-15_HopAO1-hrpA-inocu_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Underwood_2-15_HopAO1-hrpA-inocu_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: HopAO1
Stock Code:
Genetic Background: Col-5
Growth Conditions:
stratificationStratified 3 days at 4degC
Growth protocolArabidopsis thaliana accession Col-gl1 (Col-5) plants were grown in soil in growth chambers with a day/night cycle of 12 h/12 h, a light intensity of 100 µE, and a constant temperature of 20°C. Four-to 5-week-old plants were used for experiments.
LocationGrowth chamber
Growth substratecommercial soil
Average humidity80%
Average temperature20oC
Developmental Stage:
Growth Stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.70
Genetic Variation: T-DNA gene knockout
Tissue: Leaves
in vivo Treatment:
treatmentPlants were inoculated by vacuum-infiltration with a 1x10e8 cfu/ml suspension of the Pst DC3000 hrpA mutant in sterile water containing .004% Silwet L-77. Leaf tissue from at least 15 plants was collected 7 hours after inoculation for RNA isolation.
Additional Organism Information:
allele knocked outHopAO1 from Pst DC3000
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.441955
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.96
NoiseAvg:3.01,Stdev:0.10,Max:3.4,Min:2.8
BackgroundAvg:89.63,Stdev:0.55,Max:90.8,Min:88.5
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.441955
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Underwood_2-16_HopAO1-C378S-hrpA-inocu_Rep1_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Underwood_2-16_HopAO1-C378S-hrpA-inocu_Rep1_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Underwood_2-16_HopAO1-C378S-hrpA-inocu_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: HopAO1
Stock Code:
Genetic Background: Col-5
Growth Conditions:
stratificationStratified 3 days at 4degC
Growth protocolArabidopsis thaliana accession Col-gl1 (Col-5) plants were grown in soil in growth chambers with a day/night cycle of 12 h/12 h, a light intensity of 100 µE, and a constant temperature of 20°C. Four-to 5-week-old plants were used for experiments.
LocationGrowth chamber
Growth substratecommercial soil
Average humidity80%
Average temperature20oC
Developmental Stage:
Growth Stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.70
Genetic Variation: T-DNA gene knockout
Tissue: Leaves
in vivo Treatment:
treatmentTotal RNA was extracted from 1g of frozen leaf tissue using the Promega RNAgents kit according to the manufacturers protocol. Isolated RNA was further purified prior to labeling using the Qiagen RNeasy spin column kit according to the manufacturers protocol.
Additional Organism Information:
allele knocked outHopAO1 C378S from Pst DC3000
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.49446
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ3.34
NoiseAvg:3.46,Stdev:0.13,Max:4.1,Min:3.1
BackgroundAvg:106.24,Stdev:0.85,Max:107.9,Min:103.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.494460
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Underwood_2-17_HopAO1-C378S-hrpA-inocu_Rep2_ATH1

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Underwood_2-17_HopAO1-C378S-hrpA-inocu_Rep2_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Underwood_2-17_HopAO1-C378S-hrpA-inocu_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: HopAO1
Stock Code:
Genetic Background: Col-5
Growth Conditions:
stratificationStratified 3 days at 4degC
Growth protocolArabidopsis thaliana accession Col-gl1 (Col-5) plants were grown in soil in growth chambers with a day/night cycle of 12 h/12 h, a light intensity of 100 µE, and a constant temperature of 20°C. Four-to 5-week-old plants were used for experiments.
LocationGrowth chamber
Growth substratecommercial soil
Average humidity80%
Average temperature20oC
Developmental Stage:
Growth Stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.70
Genetic Variation: T-DNA gene knockout
Tissue: Leaves
in vivo Treatment:
treatmentTotal RNA was extracted from 1g of frozen leaf tissue using the Promega RNAgents kit according to the manufacturers protocol. Isolated RNA was further purified prior to labeling using the Qiagen RNeasy spin column kit according to the manufacturers protocol.
Additional Organism Information:
allele knocked outHopAO1 C378S from Pst DC3000
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.913372
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.94
NoiseAvg:2.76,Stdev:0.12,Max:3.2,Min:2.5
BackgroundAvg:91.66,Stdev:1.03,Max:94.9,Min:89.1
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.913372
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Underwood_2-18_HopAO1-C378S-hrpA-inocu_Rep3_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Underwood_2-18_HopAO1-C378S-hrpA-inocu_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Underwood_2-18_HopAO1-C378S-hrpA-inocu_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: HopAO1
Stock Code:
Genetic Background: Col-5
Growth Conditions:
stratificationStratified 3 days at 4degC
Growth protocolArabidopsis thaliana accession Col-gl1 (Col-5) plants were grown in soil in growth chambers with a day/night cycle of 12 h/12 h, a light intensity of 100 µE, and a constant temperature of 20°C. Four-to 5-week-old plants were used for experiments.
LocationGrowth chamber
Growth substratecommercial soil
Average humidity80%
Average temperature20oC
Developmental Stage:
Growth Stage(Source: Boyes et al., Plant Cell 2001 13, 1499)3.70
Genetic Variation: T-DNA gene knockout
Tissue: Leaves
in vivo Treatment:
treatmentTotal RNA was extracted from 1g of frozen leaf tissue using the Promega RNAgents kit according to the manufacturers protocol. Isolated RNA was further purified prior to labeling using the Qiagen RNeasy spin column kit according to the manufacturers protocol.
Additional Organism Information:
allele knocked outHopAO1 C378S from Pst DC3000
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.675319
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
RawQ2.63
NoiseAvg:2.56,Stdev:0.10,Max:2.9,Min:2.3
BackgroundAvg:85.19,Stdev:0.60,Max:86.9,Min:84.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.675319
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015


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