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Experiment: Wt vs gonst1-2 seedling

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-544

Aim of this experiment was to compare aerial tissue from 14d old seedlings of Wt (Col-0) vs gonst1-2 (SALK_043593) knockout plant. This will also be used to compare to a previous microarray experiment using callus with the same genotype.

About the Experimenter

Name: Jenny Mortimer
Head of Lab Name: Paul Dupree
Lab:
Address:Department of Biochemistry, University of Cambridge
Tennis Court Road, Cambridge, Cambridgeshire, CB2 1QW, UNITED KINGDOM
Postcode:
Country:
 

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
Number of Slides:6
 
Experimental Parameters:
parametergene_knock_out
Quality Control Measures Taken:
References:
 
Other Information:

Slides in this Experiment

Hybridisation Set: Mortimer: Wt vs gonst1-2 seedling

Slide: Mortimer_544-1_WT_14d_aerial-tissue_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 ("Mortimer_544-1_WT_14d_aerial-tissue_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: Mortimer_544-1_WT_14d_aerial-tissue_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Genetic Background: Col-0 (Columbia, N60000)
Age: 14
Growth Conditions:
Stratification48h, 4C, dark
SterilisationSeeds were shaken in 70% ethanol for 1 minute. This was removed and replaced with 1ml of sterilising solution (household bleach (30% v/v); Triton X-100 (0.01% v/v)) for 10 minutes. Seeds were then rinsed in 5x changes of sterile distilled water.
ProtocolFollowing stratification, seeds were grown on vertical agar plates (see below for recipe) in continuous light for 14d. Growth chamber conditions were: 20°C, 100 umol/m2/sec, 60% humidity. 2.2g/L MS salts including Vitamins ( Sigma M 5519) Agar at 0.8% 1% sucrose 0.5g/L MES Adjust to pH 5.8 using KOH
Percentage Agrose0.8
Substrate Sterilising ProcedureAutoclaved
Plant Spacing20 per plate (plate was 125 x 125 mm)
Temperature20 °C average, 20 °C day, 20 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1% sucrose pH adjusted to 5.8
Lighting(Source: Cool white fluorescent manufactured by Sylvania (3 white lights, 1 gro-lux per shelf of plants). Intensity: 100 umol/m2/secµEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.02
Tissue: shoot
Additional Organism Information:
Sample DescriptionThe seedlings were harvested 14d after transfer to the light. The plant was Col-0 WT
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:0.879122
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:
Spike_AFFX-r2-P1-cre_3-5-ratio1.252172
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.815946
Spike_AFFX-r2-Bs-dap_5_signal54.802395
NoiseAvg:1.81,Std:0.06,Min:1.6,Max:2.1
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal36.863293
#P14697
Spike_AFFX-r2-Bs-phe_M_signal38.981602
Corner-Avg:7253,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal119.903099
Spike_AFFX-r2-Ec-bioB_3_signal80.273506
Spike_AFFX-r2-Bs-lys_M_signal14.889104
Spike_AFFX-r2-P1-cre_3_signal6475.790527
Spike_AFFX-r2-Bs-lys_3-5-ratio1.675622
Spike_AFFX-r2-Bs-dap_M_signal152.830017
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio3.010571
Spike_AFFX-r2-Ec-bioB_avg-signal99.519157
Spike_AFFX-r2-Bs-thr_avg-signal76.325340
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1059.281738
Spike_AFFX-r2-Bs-phe_5_signal44.188080
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1532.628540
RawQ1.885060
Spike_AFFX-r2-Bs-lys_5_signal26.567057
Signal(A)4.235561
%A33.818501
Signal(All)148.096832
Corner+Avg:82,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal362.795624
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal49.761539
Spike_AFFX-r2-Ec-bioD_avg-signal1295.955078
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P64.432266
Spike_AFFX-r2-Bs-lys_avg-signal28.657503
Spike_AFFX-r2-P1-cre_avg-signal5823.717285
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.749233
Spike_AFFX-r2-Bs-thr_3_signal110.979546
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal44.310406
Spike_AFFX-r2-Bs-dap_3_signal222.703857
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.446856
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal371.104156
#M399
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal44.516354
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio0.977611
Spike_AFFX-r2-Bs-thr_M_signal81.133171
Signal(P)227.221268
Spike_AFFX-r2-Bs-phe_3-5-ratio1.126130
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:4299,Count:9
Spike_AFFX-r2-P1-cre_5_signal5171.644043
Spike_AFFX-r2-Bs-phe_M_detectionP
#A7714
Signal(M)14.898472
BackgroundAvg:46.20,Std:0.29,Min:45.7,Max:47.0
Spike_AFFX-r2-Ec-bioC_avg-signal366.949890
Spike_AFFX-r2-Bs-dap_avg-signal143.445419
Spike_AFFX-r2-Bs-dap_3-5-ratio4.063761
Spike_AFFX-r2-Ec-bioB_5_signal98.380852
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.879122
NF1.000000
HZ4
Tau0.015000

Slide: Mortimer_544-2_WT_14d_aerial-tissue_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 ("Mortimer_544-2_WT_14d_aerial-tissue_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: Mortimer_544-2_WT_14d_aerial-tissue_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Genetic Background: Col-0 (Columbia, N60000)
Age: 14
Growth Conditions:
Stratification48h, 4C, dark
SterilisationSeeds were shaken in 70% ethanol for 1 minute. This was removed and replaced with 1ml of sterilising solution (household bleach (30% v/v); Triton X-100 (0.01% v/v)) for 10 minutes. Seeds were then rinsed in 5x changes of sterile distilled water.
ProtocolFollowing stratification, seeds were grown on vertical agar plates (see below for recipe) in continuous light for 14d. Growth chamber conditions were: 20°C, 100 umol/m2/sec, 60% humidity. 2.2g/L MS salts including Vitamins ( Sigma M 5519) Agar at 0.8% 1% sucrose 0.5g/L MES Adjust to pH 5.8 using KOH
Percentage Agrose0.8
Substrate Sterilising ProcedureAutoclaved
Plant Spacing20 per plate (plate was 125 x 125 mm)
Temperature20 °C average, 20 °C day, 20 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1% sucrose pH adjusted to 5.8
Lighting(Source: Cool white fluorescent manufactured by Sylvania (3 white lights, 1 gro-lux per shelf of plants). Intensity: 100 umol/m2/secµEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.02
Tissue: shoot
Additional Organism Information:
Sample DescriptionThe seedlings were harvested 14d after transfer to the light. The plant is Col-0 wild type.
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:0.836417
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:
Spike_AFFX-r2-P1-cre_3-5-ratio0.976953
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.786943
Spike_AFFX-r2-Bs-dap_5_signal46.417114
NoiseAvg:2.14,Std:0.07,Min:1.9,Max:2.3
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal26.347273
#P14830
Spike_AFFX-r2-Bs-phe_M_signal29.548418
Corner-Avg:8060,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal104.950935
Spike_AFFX-r2-Ec-bioB_3_signal67.008995
Spike_AFFX-r2-Bs-lys_M_signal16.058060
Spike_AFFX-r2-P1-cre_3_signal4008.805176
Spike_AFFX-r2-Bs-lys_3-5-ratio2.115113
Spike_AFFX-r2-Bs-dap_M_signal105.201996
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio2.722260
Spike_AFFX-r2-Ec-bioB_avg-signal85.703667
Spike_AFFX-r2-Bs-thr_avg-signal55.007309
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal764.107849
Spike_AFFX-r2-Bs-phe_5_signal29.180918
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1358.431274
RawQ2.203553
Spike_AFFX-r2-Bs-lys_5_signal14.998463
Signal(A)4.457851
%A33.345024
Signal(All)145.210632
Corner+Avg:87,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal304.996094
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal42.645130
Spike_AFFX-r2-Ec-bioD_avg-signal1061.269531
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P65.015343
Spike_AFFX-r2-Bs-lys_avg-signal20.926657
Spike_AFFX-r2-P1-cre_avg-signal4056.090576
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.639632
Spike_AFFX-r2-Bs-thr_3_signal71.724121
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal33.791489
Spike_AFFX-r2-Bs-dap_3_signal182.768143
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.777800
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal280.314575
#M374
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal31.723452
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.088049
Spike_AFFX-r2-Bs-thr_M_signal66.950523
Signal(P)220.639954
Spike_AFFX-r2-Bs-phe_3-5-ratio1.461405
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:5572,Count:9
Spike_AFFX-r2-P1-cre_5_signal4103.375977
Spike_AFFX-r2-Bs-phe_M_detectionP
#A7606
Signal(M)16.731827
BackgroundAvg:55.44,Std:0.77,Min:53.1,Max:57.7
Spike_AFFX-r2-Ec-bioC_avg-signal292.655334
Spike_AFFX-r2-Bs-dap_avg-signal111.462425
Spike_AFFX-r2-Bs-dap_3-5-ratio3.937516
Spike_AFFX-r2-Ec-bioB_5_signal85.151062
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.836417
NF1.000000
HZ4
Tau0.015000

Slide: Mortimer_544-3_WT_14d_aerial-tissue_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 ("Mortimer_544-3_WT_14d_aerial-tissue_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: Mortimer_544-3_WT_14d_aerial-tissue_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Genetic Background: Col-0 (Columbia, N60000)
Age: 14
Growth Conditions:
Stratification48h, 4C, dark
SterilisationSeeds were shaken in 70% ethanol for 1 minute. This was removed and replaced with 1ml of sterilising solution (household bleach (30% v/v); Triton X-100 (0.01% v/v)) for 10 minutes. Seeds were then rinsed in 5x changes of sterile distilled water.
ProtocolFollowing stratification, seeds were grown on vertical agar plates (see below for recipe) in continuous light for 14d. Growth chamber conditions were: 20°C, 100 umol/m2/sec, 60% humidity. 2.2g/L MS salts including Vitamins ( Sigma M 5519) Agar at 0.8% 1% sucrose 0.5g/L MES Adjust to pH 5.8 using KOH
Percentage Agrose0.8
Substrate Sterilising ProcedureAutoclaved
Plant Spacing20 per plate (plate was 125 x 125 mm)
Temperature20 °C average, 20 °C day, 20 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1% sucrose pH adjusted to 5.8
Lighting(Source: Cool white fluorescent manufactured by Sylvania (3 white lights, 1 gro-lux per shelf of plants). Intensity: 100 umol/m2/secµEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.02
Tissue: shoot
Additional Organism Information:
Sample DescriptionThe seedlings were harvested 14d after transfer to the light. The plant is Col-0 wild type.
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:1.338164
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:
Spike_AFFX-r2-P1-cre_3-5-ratio1.188886
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.892523
Spike_AFFX-r2-Bs-dap_5_signal35.310902
NoiseAvg:1.62,Std:0.08,Min:1.5,Max:1.8
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal25.704170
#P13805
Spike_AFFX-r2-Bs-phe_M_signal35.644718
Corner-Avg:7365,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal115.809677
Spike_AFFX-r2-Ec-bioB_3_signal82.478836
Spike_AFFX-r2-Bs-lys_M_signal10.562810
Spike_AFFX-r2-P1-cre_3_signal7679.050293
Spike_AFFX-r2-Bs-lys_3-5-ratio1.399734
Spike_AFFX-r2-Bs-dap_M_signal91.040604
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio3.976402
Spike_AFFX-r2-Ec-bioB_avg-signal96.899788
Spike_AFFX-r2-Bs-thr_avg-signal60.359692
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1215.370605
Spike_AFFX-r2-Bs-phe_5_signal34.480347
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1929.011963
RawQ1.775720
Spike_AFFX-r2-Bs-lys_5_signal19.440935
Signal(A)5.593184
%A37.575626
Signal(All)160.296371
Corner+Avg:77,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal420.495270
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal28.965990
Spike_AFFX-r2-Ec-bioD_avg-signal1572.191284
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P60.521702
Spike_AFFX-r2-Bs-lys_avg-signal19.071962
Spike_AFFX-r2-P1-cre_avg-signal7069.041016
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.902674
Spike_AFFX-r2-Bs-thr_3_signal102.210114
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal33.030354
Spike_AFFX-r2-Bs-dap_3_signal130.900925
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.587180
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal413.669769
#M434
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal27.212145
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.016500
Spike_AFFX-r2-Bs-thr_M_signal53.164803
Signal(P)260.721619
Spike_AFFX-r2-Bs-phe_3-5-ratio0.840072
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:4288,Count:9
Spike_AFFX-r2-P1-cre_5_signal6459.031738
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8571
Signal(M)21.104465
BackgroundAvg:67.89,Std:0.57,Min:66.9,Max:69.5
Spike_AFFX-r2-Ec-bioC_avg-signal417.082520
Spike_AFFX-r2-Bs-dap_avg-signal85.750816
Spike_AFFX-r2-Bs-dap_3-5-ratio3.707097
Spike_AFFX-r2-Ec-bioB_5_signal92.410851
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF1.338164
NF1.000000
HZ4
Tau0.015000

Slide: Mortimer_544-4_gonst1-2 14d_aerial-tissue_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 ("Mortimer_544-4_gonst1-2 14d_aerial-tissue_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: Mortimer_544-4_gonst1-2 14d_aerial-tissue_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: SALK_043593 (I) (AI)
Stock Code: N543593
Genetic Background: Col-0 (Columbia, N60000)
Age: 14
Growth Conditions:
Stratification48h, 4C, dark
SterilisationSeeds were shaken in 70% ethanol for 1 minute. This was removed and replaced with 1ml of sterilising solution (household bleach (30% v/v); Triton X-100 (0.01% v/v)) for 10 minutes. Seeds were then rinsed in 5x changes of sterile distilled water.
ProtocolFollowing stratification, seeds were grown on vertical agar plates (see below for recipe) in continuous light for 14d. Growth chamber conditions were: 20°C, 100 umol/m2/sec, 60% humidity. 2.2g/L MS salts including Vitamins ( Sigma M 5519) Agar at 0.8% 1% sucrose 0.5g/L MES Adjust to pH 5.8 using KOH
Percentage Agrose0.8
Substrate Sterilising ProcedureAutoclaved
Plant Spacing20 per plate (plate was 125 x 125 mm)
Temperature20 °C average, 20 °C day, 20 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1% sucrose pH adjusted to 5.8
Lighting(Source: Cool white fluorescent manufactured by Sylvania (3 white lights, 1 gro-lux per shelf of plants). Intensity: 100 umol/m2/secµEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.02
Genetic Variation: T-DNA insertion
Tissue: shoot
Additional Organism Information:
Sample DescriptionThe seedlings were harvested 14d after transfer to the light. The plant was a homozygous knockout of the gonst 1 gene (At2g13650).
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:0.566692
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:
Spike_AFFX-r2-P1-cre_3-5-ratio1.174315
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.054939
Spike_AFFX-r2-Bs-dap_5_signal57.286911
NoiseAvg:2.14,Std:0.06,Min:1.9,Max:2.3
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal28.214355
#P15256
Spike_AFFX-r2-Bs-phe_M_signal34.843506
Corner-Avg:7331,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal91.996384
Spike_AFFX-r2-Ec-bioB_3_signal76.209709
Spike_AFFX-r2-Bs-lys_M_signal16.349705
Spike_AFFX-r2-P1-cre_3_signal4289.667969
Spike_AFFX-r2-Bs-lys_3-5-ratio2.377697
Spike_AFFX-r2-Bs-dap_M_signal120.698952
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio2.819524
Spike_AFFX-r2-Ec-bioB_avg-signal80.148987
Spike_AFFX-r2-Bs-thr_avg-signal56.935955
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal778.324524
Spike_AFFX-r2-Bs-phe_5_signal40.335976
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1179.048462
RawQ1.952128
Spike_AFFX-r2-Bs-lys_5_signal18.036604
Signal(A)3.214658
%A31.587023
Signal(All)138.088470
Corner+Avg:87,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal319.758514
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal43.244064
Spike_AFFX-r2-Ec-bioD_avg-signal978.686523
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P66.882950
Spike_AFFX-r2-Bs-lys_avg-signal25.757294
Spike_AFFX-r2-P1-cre_avg-signal3971.288574
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.530031
Spike_AFFX-r2-Bs-thr_3_signal79.551041
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal39.474514
Spike_AFFX-r2-Bs-dap_3_signal166.697571
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.514855
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal294.354004
#M349
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal42.885574
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.086306
Spike_AFFX-r2-Bs-thr_M_signal63.042458
Signal(P)204.652893
Spike_AFFX-r2-Bs-phe_3-5-ratio1.072097
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:5244,Count:9
Spike_AFFX-r2-P1-cre_5_signal3652.909424
Spike_AFFX-r2-Bs-phe_M_detectionP
#A7205
Signal(M)12.756314
BackgroundAvg:73.54,Std:0.21,Min:72.9,Max:74.2
Spike_AFFX-r2-Ec-bioC_avg-signal307.056274
Spike_AFFX-r2-Bs-dap_avg-signal114.894470
Spike_AFFX-r2-Bs-dap_3-5-ratio2.909872
Spike_AFFX-r2-Ec-bioB_5_signal72.240883
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.566692
NF1.000000
HZ4
Tau0.015000

Slide: Mortimer_544-5_gonst1-2 14d_aerial-tissue_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 ("Mortimer_544-5_gonst1-2 14d_aerial-tissue_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: Mortimer_544-5_gonst1-2 14d_aerial-tissue_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: SALK_043593 (I) (AI)
Stock Code: N543593
Genetic Background: Col-0 (Columbia, N60000)
Age: 14
Growth Conditions:
Stratification48h, 4C, dark
SterilisationSeeds were shaken in 70% ethanol for 1 minute. This was removed and replaced with 1ml of sterilising solution (household bleach (30% v/v); Triton X-100 (0.01% v/v)) for 10 minutes. Seeds were then rinsed in 5x changes of sterile distilled water.
ProtocolFollowing stratification, seeds were grown on vertical agar plates (see below for recipe) in continuous light for 14d. Growth chamber conditions were: 20°C, 100 umol/m2/sec, 60% humidity. 2.2g/L MS salts including Vitamins ( Sigma M 5519) Agar at 0.8% 1% sucrose 0.5g/L MES Adjust to pH 5.8 using KOH
Percentage Agrose0.8
Substrate Sterilising ProcedureAutoclaved
Plant Spacing20 per plate (plate was 125 x 125 mm)
Temperature20 °C average, 20 °C day, 20 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1% sucrose pH adjusted to 5.8
Lighting(Source: Cool white fluorescent manufactured by Sylvania (3 white lights, 1 gro-lux per shelf of plants). Intensity: 100 umol/m2/secµEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.02
Genetic Variation: T-DNA insertion
Tissue: shoot
Additional Organism Information:
Sample DescriptionThe seedlings were harvested 14d after transfer to the light. The plant was a homozygous knockout of the gonst 1 gene (At2g13650).
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:0.882914
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:
Spike_AFFX-r2-P1-cre_3-5-ratio1.331353
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.206678
Spike_AFFX-r2-Bs-dap_5_signal54.587227
NoiseAvg:1.93,Std:0.09,Min:1.8,Max:2.2
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal39.348602
#P14548
Spike_AFFX-r2-Bs-phe_M_signal31.480715
Corner-Avg:7624,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal127.078346
Spike_AFFX-r2-Ec-bioB_3_signal111.528419
Spike_AFFX-r2-Bs-lys_M_signal11.731617
Spike_AFFX-r2-P1-cre_3_signal7112.488281
Spike_AFFX-r2-Bs-lys_3-5-ratio1.091016
Spike_AFFX-r2-Bs-dap_M_signal151.197357
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio2.297236
Spike_AFFX-r2-Ec-bioB_avg-signal110.344261
Spike_AFFX-r2-Bs-thr_avg-signal66.440773
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1331.221802
Spike_AFFX-r2-Bs-phe_5_signal33.234917
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal2036.462891
RawQ2.075298
Spike_AFFX-r2-Bs-lys_5_signal17.373198
Signal(A)4.266199
%A34.568172
Signal(All)147.620041
Corner+Avg:81,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal440.084015
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal34.905899
Spike_AFFX-r2-Ec-bioD_avg-signal1683.842285
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P63.779045
Spike_AFFX-r2-Bs-lys_avg-signal16.019751
Spike_AFFX-r2-P1-cre_avg-signal6227.394531
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.652784
Spike_AFFX-r2-Bs-thr_3_signal90.393036
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal33.207176
Spike_AFFX-r2-Bs-dap_3_signal174.481506
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.529770
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal430.835297
#M377
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal18.954439
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.021467
Spike_AFFX-r2-Bs-thr_M_signal69.580681
Signal(P)228.711517
Spike_AFFX-r2-Bs-phe_3-5-ratio1.050278
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:4840,Count:9
Spike_AFFX-r2-P1-cre_5_signal5342.300781
Spike_AFFX-r2-Bs-phe_M_detectionP
#A7885
Signal(M)16.654783
BackgroundAvg:74.66,Std:0.59,Min:73.2,Max:76.2
Spike_AFFX-r2-Ec-bioC_avg-signal435.459656
Spike_AFFX-r2-Bs-dap_avg-signal126.755363
Spike_AFFX-r2-Bs-dap_3-5-ratio3.196380
Spike_AFFX-r2-Ec-bioB_5_signal92.426018
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.882914
NF1.000000
HZ4
Tau0.015000

Slide: Mortimer_544-6_gonst1-2 14d_aerial-tissue_Rep3_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 ("Mortimer_544-6_gonst1-2 14d_aerial-tissue_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: Mortimer_544-6_gonst1-2 14d_aerial-tissue_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: SALK_043593 (I) (AI)
Stock Code: N543593
Genetic Background: Col-0 (Columbia, N60000)
Age: 14
Growth Conditions:
Stratification48h, 4C, dark
SterilisationSeeds were shaken in 70% ethanol for 1 minute. This was removed and replaced with 1ml of sterilising solution (household bleach (30% v/v); Triton X-100 (0.01% v/v)) for 10 minutes. Seeds were then rinsed in 5x changes of sterile distilled water.
ProtocolFollowing stratification, seeds were grown on vertical agar plates (see below for recipe) in continuous light for 14d. Growth chamber conditions were: 20°C, 100 umol/m2/sec, 60% humidity. 2.2g/L MS salts including Vitamins ( Sigma M 5519) Agar at 0.8% 1% sucrose 0.5g/L MES Adjust to pH 5.8 using KOH
Percentage Agrose0.8
Substrate Sterilising ProcedureAutoclaved
Plant Spacing20 per plate (plate was 125 x 125 mm)
Temperature20 °C average, 20 °C day, 20 °C night
Humidity60 % average, 60 % day, 60 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1% sucrose pH adjusted to 5.8
Lighting(Source: Cool white fluorescent manufactured by Sylvania (3 white lights, 1 gro-lux per shelf of plants). Intensity: 100 umol/m2/secµEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.02
Genetic Variation: T-DNA insertion
Tissue: shoot
Additional Organism Information:
Sample DescriptionThe seedlings were harvested 14d after transfer to the light. The plant was a homozygous knockout of the gonst 1 gene (At2g13650).
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:0.903288
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:
Spike_AFFX-r2-P1-cre_3-5-ratio1.400145
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.780213
Spike_AFFX-r2-Bs-dap_5_signal67.711632
NoiseAvg:2.00,Std:0.07,Min:1.7,Max:2.1
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal26.680138
#P14572
Spike_AFFX-r2-Bs-phe_M_signal26.272762
Corner-Avg:6778,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal94.812698
Spike_AFFX-r2-Ec-bioB_3_signal56.768009
Spike_AFFX-r2-Bs-lys_M_signal12.284362
Spike_AFFX-r2-P1-cre_3_signal5689.395020
Spike_AFFX-r2-Bs-lys_3-5-ratio1.816694
Spike_AFFX-r2-Bs-dap_M_signal92.162674
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio3.209432
Spike_AFFX-r2-Ec-bioB_avg-signal74.780121
Spike_AFFX-r2-Bs-thr_avg-signal56.554169
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal848.360229
Spike_AFFX-r2-Bs-phe_5_signal30.375256
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1257.177979
RawQ2.035554
Spike_AFFX-r2-Bs-lys_5_signal10.480249
Signal(A)5.099171
%A34.445419
Signal(All)144.172440
Corner+Avg:72,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal399.723846
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal29.400520
Spike_AFFX-r2-Ec-bioD_avg-signal1052.769043
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P63.884262
Spike_AFFX-r2-Bs-lys_avg-signal13.934672
Spike_AFFX-r2-P1-cre_avg-signal4876.413086
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.670320
Spike_AFFX-r2-Bs-thr_3_signal85.628098
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal28.682846
Spike_AFFX-r2-Bs-dap_3_signal144.817337
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.481892
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal301.081268
#M381
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal19.039404
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.327628
Spike_AFFX-r2-Bs-thr_M_signal57.354259
Signal(P)222.445587
Spike_AFFX-r2-Bs-phe_3-5-ratio0.967910
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:4968,Count:9
Spike_AFFX-r2-P1-cre_5_signal4063.431641
Spike_AFFX-r2-Bs-phe_M_detectionP
#A7857
Signal(M)18.457260
BackgroundAvg:76.84,Std:0.61,Min:75.2,Max:78.4
Spike_AFFX-r2-Ec-bioC_avg-signal350.402557
Spike_AFFX-r2-Bs-dap_avg-signal101.563881
Spike_AFFX-r2-Bs-dap_3-5-ratio2.138736
Spike_AFFX-r2-Ec-bioB_5_signal72.759666
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.903288
NF1.000000
HZ4
Tau0.015000


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