NASC's International Affymetrix Service

AI logo

NASCArrays

home, selection, spot history, gene swinger, two gene scatterplot, digital northern, bulk gene download, tutorial, help, Affymetrix site, NASC

Experiment: Determining the downstream genetic targets of the transcription factor AtGLK1

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-438

AimTo discover the target genes of Golden2-like transcription factors in Arabidopsis.BackgroundThe Golden2-like (GLK) family of transcription factors is essential for proper chloroplast development in several plant species. In Arabidopsis, there are two largely redundant GLK genes, GLK1 and GLK2. Double mutant plants are pale green and reduced in stature. Their most notable chloroplast-related phenotype is impaired thylakoid membrane appression and reduction in steady-state levels of Photosystem II components. GLK proteins group within the GARP family of myb transcription factors; however, the genes they regulate remain unknown.Experimental DesignWe have employed a two-component dexamethasone-inducible expression system [1]. An overexpressed fusion protein comprising the glucocorticoid receptor (GR), the lac repressor and the Gal4 activation domain resides in the plant cytoplasm. This protein, termed LHGR-N, enters the nucleus once dexamethasone binds to the GR domain. It then activates transcription from the lac operator promoter sequences present on separate transgenes which carry the cDNA to be induced. Since overexpression of either GLK1 or GLK2 is sufficient to complement the mutant phenotype, we reasoned that induction of expression of GLK1 and GLK2 cDNAs in the glk1;glk2 double mutant background should allow determination of their downstream genetic targets. In the first case, we decided to focus on GLK1 only.Genetic linesPlants carrying a single copy of the LHGR-N transgene, conferring kanamycin resistance, were crossed with glk1;glk2 double mutant plants. A single homozygous F3 line was selected on the basis of pale green mutant phenotype and kanamycin resistance. This line was transformed with a construct carrying the GLK1 cDNA downstream of six repeats of the lac operator (pOp6) and a single CaMV minimal promoter. This construct confers hygromycin resistance. Transformants were confirmed by PCR and southern blot, before being selected on the basis of transcript accumulation following induction by dexamethasone. The line with the strongest induction response was chosen for microarray experiments.Tissues: Approx. 100 10-day old seedlings were grown on MS plates and transferred to 100 ml liquid MS for two further days prior to induction. Whole seedlings were harvested.Treatment: either a) 10 uM dexamethasone dissolved in DMSO (INDUCED samples) or b) 0.1% v/v DMSO (CONTROL samples). Samples were harvested 4 and 24 hours post-treatment. Separate vessels were used for each time point and treatment. Two complete replicates were performed.Reference1. Craft, J. et al. (2005) Plant J. 41 899-918.

About the Experimenter

Name:Dr Mark Waters
Head of Lab Name:Prof Jane Langdale
Lab:
Address:Department of Plant Sciences
University of Oxford
South Parks Road
OXFORD
Postcode: OX1 3RB
Country: UK
 
Telephone Number: 01865275030

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: compound_based_treatment
Number of Slides:10
 
Experimental Parameters: gene_knock_in
parameter
Quality Control Measures Taken:
no-plants-pooled
References:
 
Other Information:

Slides in this Experiment

Hybridisation Set: Waters: Determining the downstream genetic targets of the transcription factor AtGLK1_genome

Slide: Waters_1-1_0hr-Control_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 ("Waters_1-1_0hr-Control_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: Waters_1-1_0hr-Control_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Atglk1;Atglk2
Stock Code:
Genetic Background: Col-0
Age: 0hr timepoint
Growth Conditions:
StratificationMS agar plates containing sterilised seed were placed in the dark at 4°C for three days.
SterilisationSeed sterilisation was performed in 15 ml Falcon tubes. Seeds were washed in 70% ethanol for 5 minutes with shaking. The tubes were centrifuged and the ethanol replaced with freshly-prepared 50% sodium hypochlorite solution (BDH; available chlorine %), and the tubes were shaken for 10 minutes. The seeds were pelleted by centrifugation, the bleach solution replaced with sterile pure water, and the tubes shaken vigorously. The seeds were pelleted again and the rinsing was repeated twice more.
OtherTo spread the seed evenly on MS agar plates, the sterilised seeds were resuspended in sterile 0.1% agar and transferred by pipette on to 10 cm square plates. The plates were shaken gently to disperse the seed/agar suspension.
LocationGrowth Room
Growth substrate typeAgar 0.7%
Agar SterilisationAgar was added to a concentration of 0.7% (w/v) agar and the medium was autoclaved at 121 deg. C for 20 mins. The medium was allowed to cool to 50 deg. C. and hygromycin B was added to 20 µg/ml immediately before pouring into 10 cm square plates.
Spacing~200 seeds/plate
Average temperature20oC
Average humidity50%
Growth mediumMurashige & Skoog basal salt mixture
Growth medium modifications1x Gamborg's vitamins and 1.5% (w/v) sucrose added and pH adjusted to 5.9. 20 µg/ml hygromycin B added after sterilisation
Growth protocolSeed was surface-sterilised and spread on MS plates supplemented with 1.5% (w/v) sucrose, 0.7% (w/v) agar, 1x Gamborg's vitamins and 20 µg/ml hygromycin B. The plates were sealed, stratified for three days and transferred to an artificial growth room held at a constant 20°C, with a 16h/8h light/dark cycle. Light was provided by fluorescent tubes, generating 85 µmol/m2/s PAR. After ten days, seedlings were transferred from the plates into liquid MS media in a sterile Petri dish, in order to separate the seedlings. Using forceps, individual plants of a similar growth stage (first pair of leaves > 1 mm or Stage 1.02 as defined by Boyes et al., 2001) were selected and placed into 100 ml liquid MS medium in a 250 ml conical flask. Approximately 100 seedlings were transferred, and the flask was sealed with a foam bung. The flask was returned to the growth room and shaken at 76 rpm to provide aeration and adequate mixing. After 48 hours, the seedlings were harvested by filtering through one layer of cheesecloth, and quickly dabbing dry between two pieces of 3MM chromoatography paper. The seedlings were then flash-frozen in liquid nitrogen.
Genetic Variation: transposon gene knock out
Other Information:
AlleleAt2g20570
AlleleAt5g44190

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:0.427527993917
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:
RawQ2.70
NoiseAvg:3.14,Stdev:0.09,Max:3.5,Min:2.9
Central-Avg:6847,Count:9
Corner+Avg:101,Count:32
Corner-Avg:9248,Count:32
BackgroundAvg:68.17,Stdev:1.02,Max:70.9,Min:65.1
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.427527993917
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Waters_1-2_0hr-control_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 ("Waters_1-2_0hr-control_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: Waters_1-2_0hr-control_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Atglk1;Atglk2
Stock Code:
Genetic Background: Col-0
Age: 0hr timepoint
Growth Conditions:
StratificationMS agar plates containing sterilised seed were placed in the dark at 4°C for three days.
SterilisationSeed sterilisation was performed in 15 ml Falcon tubes. Seeds were washed in 70% ethanol for 5 minutes with shaking. The tubes were centrifuged and the ethanol replaced with freshly-prepared 50% sodium hypochlorite solution (BDH; available chlorine %), and the tubes were shaken for 10 minutes. The seeds were pelleted by centrifugation, the bleach solution replaced with sterile pure water, and the tubes shaken vigorously. The seeds were pelleted again and the rinsing was repeated twice more.
OtherTo spread the seed evenly on MS agar plates, the sterilised seeds were resuspended in sterile 0.1% agar and transferred by pipette on to 10 cm square plates. The plates were shaken gently to disperse the seed/agar suspension.
LocationGrowth Room
Growth substrate typeAgar 0.7%
Agar SterilisationAgar was added to a concentration of 0.7% (w/v) agar and the medium was autoclaved at 121 deg. C for 20 mins. The medium was allowed to cool to 50 deg. C. and hygromycin B was added to 20 µg/ml immediately before pouring into 10 cm square plates.
Spacing~200 seeds/plate
Average temperature20oC
Average humidity50%
Growth mediumMurashige & Skoog basal salt mixture
Growth medium modifications1x Gamborg's vitamins and 1.5% (w/v) sucrose added and pH adjusted to 5.9. 20 µg/ml hygromycin B added after sterilisation
Growth protocolSeed was surface-sterilised and spread on MS plates supplemented with 1.5% (w/v) sucrose, 0.7% (w/v) agar, 1x Gamborg's vitamins and 20 µg/ml hygromycin B. The plates were sealed, stratified for three days and transferred to an artificial growth room held at a constant 20°C, with a 16h/8h light/dark cycle. Light was provided by fluorescent tubes, generating 85 µmol/m2/s PAR. After ten days, seedlings were transferred from the plates into liquid MS media in a sterile Petri dish, in order to separate the seedlings. Using forceps, individual plants of a similar growth stage (first pair of leaves > 1 mm or Stage 1.02 as defined by Boyes et al., 2001) were selected and placed into 100 ml liquid MS medium in a 250 ml conical flask. Approximately 100 seedlings were transferred, and the flask was sealed with a foam bung. The flask was returned to the growth room and shaken at 76 rpm to provide aeration and adequate mixing. After 48 hours, the seedlings were harvested by filtering through one layer of cheesecloth, and quickly dabbing dry between two pieces of 3MM chromoatography paper. The seedlings were then flash-frozen in liquid nitrogen.
Genetic Variation: transposon gene knock out
Other Information:
AlleleAt2g20570
AlleleAt5g44190

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:0.825338065624
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:
RawQ2.61
NoiseAvg:2.71,Stdev:0.07,Max:2.9,Min:2.5
Central-Avg:5564,Count:9
Corner+Avg:79,Count:32
Corner-Avg:8084,Count:32
BackgroundAvg:65.74,Stdev:0.57,Max:67.2,Min:64.1
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.825338065624
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Waters_1-3_4hr-Control_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 ("Waters_1-3_4hr-Control_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: Waters_1-3_4hr-Control_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Atglk1;Atglk2
Stock Code:
Genetic Background: Col-0
Age: 4hr timepoint
Growth Conditions:
StratificationMS agar plates containing sterilised seed were placed in the dark at 4°C for three days.
SterilisationSeed sterilisation was performed in 15 ml Falcon tubes. Seeds were washed in 70% ethanol for 5 minutes with shaking. The tubes were centrifuged and the ethanol replaced with freshly-prepared 50% sodium hypochlorite solution (BDH; available chlorine %), and the tubes were shaken for 10 minutes. The seeds were pelleted by centrifugation, the bleach solution replaced with sterile pure water, and the tubes shaken vigorously. The seeds were pelleted again and the rinsing was repeated twice more.
OtherTo spread the seed evenly on MS agar plates, the sterilised seeds were resuspended in sterile 0.1% agar and transferred by pipette on to 10 cm square plates. The plates were shaken gently to disperse the seed/agar suspension.
LocationGrowth Room
Growth substrate typeAgar 0.7%
Agar SterilisationAgar was added to a concentration of 0.7% (w/v) agar and the medium was autoclaved at 121 deg. C for 20 mins. The medium was allowed to cool to 50 deg. C. and hygromycin B was added to 20 µg/ml immediately before pouring into 10 cm square plates.
Spacing~200 seeds/plate
Average temperature20oC
Average humidity50%
Growth mediumMurashige & Skoog basal salt mixture
Growth medium modifications1x Gamborg's vitamins and 1.5% (w/v) sucrose added and pH adjusted to 5.9. 20 µg/ml hygromycin B added after sterilisation
Growth protocolSeed was surface-sterilised and spread on MS plates supplemented with 1.5% (w/v) sucrose, 0.7% (w/v) agar, 1x Gamborg's vitamins and 20 µg/ml hygromycin B. The plates were sealed, stratified for three days and transferred to an artificial growth room held at a constant 20°C, with a 16h/8h light/dark cycle. Light was provided by fluorescent tubes, generating 85 µmol/m2/s PAR. After ten days, seedlings were transferred from the plates into liquid MS media in a sterile Petri dish, in order to separate the seedlings. Using forceps, individual plants of a similar growth stage (first pair of leaves > 1 mm or Stage 1.02 as defined by Boyes et al., 2001) were selected and placed into 100 ml liquid MS medium in a 250 ml conical flask. Approximately 100 seedlings were transferred, and the flask was sealed with a foam bung. The flask was returned to the growth room and shaken at 76 rpm to provide aeration and adequate mixing. After 48 hours, the seedlings were harvested by filtering through one layer of cheesecloth, and quickly dabbing dry between two pieces of 3MM chromoatography paper. The seedlings were then flash-frozen in liquid nitrogen.
Genetic Variation: transposon gene knock out
Other Information:
AlleleAt2g20570
AlleleAt5g44190

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:0.674777448177
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:
RawQ2.44
NoiseAvg:2.68,Stdev:0.08,Max:2.9,Min:2.5
Central-Avg:6378,Count:9
Corner+Avg:93,Count:32
Corner-Avg:8819,Count:32
BackgroundAvg:61.65,Stdev:0.92,Max:64.1,Min:59.8
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.674777448177
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Waters_1-4_4hr-control_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 ("Waters_1-4_4hr-control_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: Waters_1-4_4hr-control_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Atglk1;Atglk2
Stock Code:
Genetic Background: Col-0
Age: 4hr timepoint
Growth Conditions:
StratificationMS agar plates containing sterilised seed were placed in the dark at 4°C for three days.
SterilisationSeed sterilisation was performed in 15 ml Falcon tubes. Seeds were washed in 70% ethanol for 5 minutes with shaking. The tubes were centrifuged and the ethanol replaced with freshly-prepared 50% sodium hypochlorite solution (BDH; available chlorine %), and the tubes were shaken for 10 minutes. The seeds were pelleted by centrifugation, the bleach solution replaced with sterile pure water, and the tubes shaken vigorously. The seeds were pelleted again and the rinsing was repeated twice more.
OtherTo spread the seed evenly on MS agar plates, the sterilised seeds were resuspended in sterile 0.1% agar and transferred by pipette on to 10 cm square plates. The plates were shaken gently to disperse the seed/agar suspension.
LocationGrowth Room
Growth substrate typeAgar 0.7%
Agar SterilisationAgar was added to a concentration of 0.7% (w/v) agar and the medium was autoclaved at 121 deg. C for 20 mins. The medium was allowed to cool to 50 deg. C. and hygromycin B was added to 20 µg/ml immediately before pouring into 10 cm square plates.
Spacing~200 seeds/plate
Average temperature20oC
Average humidity50%
Growth mediumMurashige & Skoog basal salt mixture
Growth medium modifications1x Gamborg's vitamins and 1.5% (w/v) sucrose added and pH adjusted to 5.9. 20 µg/ml hygromycin B added after sterilisation
Growth protocolSeed was surface-sterilised and spread on MS plates supplemented with 1.5% (w/v) sucrose, 0.7% (w/v) agar, 1x Gamborg's vitamins and 20 µg/ml hygromycin B. The plates were sealed, stratified for three days and transferred to an artificial growth room held at a constant 20°C, with a 16h/8h light/dark cycle. Light was provided by fluorescent tubes, generating 85 µmol/m2/s PAR. After ten days, seedlings were transferred from the plates into liquid MS media in a sterile Petri dish, in order to separate the seedlings. Using forceps, individual plants of a similar growth stage (first pair of leaves > 1 mm or Stage 1.02 as defined by Boyes et al., 2001) were selected and placed into 100 ml liquid MS medium in a 250 ml conical flask. Approximately 100 seedlings were transferred, and the flask was sealed with a foam bung. The flask was returned to the growth room and shaken at 76 rpm to provide aeration and adequate mixing. After 48 hours, the seedlings were harvested by filtering through one layer of cheesecloth, and quickly dabbing dry between two pieces of 3MM chromoatography paper. The seedlings were then flash-frozen in liquid nitrogen.
Genetic Variation: transposon gene knock out
Other Information:
AlleleAt2g20570
AlleleAt5g44190

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:1.02566754818
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:
RawQ2.13
NoiseAvg:1.98,Stdev:0.06,Max:2.1,Min:1.8
Central-Avg:5688,Count:9
Corner+Avg:62,Count:32
Corner-Avg:7297,Count:32
BackgroundAvg:48.29,Stdev:0.58,Max:50.2,Min:46.8
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF1.025667548180
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Waters_1-5_24hr-Control_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 ("Waters_1-5_24hr-Control_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: Waters_1-5_24hr-Control_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Atglk1;Atglk2
Stock Code:
Genetic Background: Col-0
Age: 24hr timepoint
Growth Conditions:
StratificationMS agar plates containing sterilised seed were placed in the dark at 4°C for three days.
SterilisationSeed sterilisation was performed in 15 ml Falcon tubes. Seeds were washed in 70% ethanol for 5 minutes with shaking. The tubes were centrifuged and the ethanol replaced with freshly-prepared 50% sodium hypochlorite solution (BDH; available chlorine %), and the tubes were shaken for 10 minutes. The seeds were pelleted by centrifugation, the bleach solution replaced with sterile pure water, and the tubes shaken vigorously. The seeds were pelleted again and the rinsing was repeated twice more.
OtherTo spread the seed evenly on MS agar plates, the sterilised seeds were resuspended in sterile 0.1% agar and transferred by pipette on to 10 cm square plates. The plates were shaken gently to disperse the seed/agar suspension.
LocationGrowth Room
Growth substrate typeAgar 0.7%
Agar SterilisationAgar was added to a concentration of 0.7% (w/v) agar and the medium was autoclaved at 121 deg. C for 20 mins. The medium was allowed to cool to 50 deg. C. and hygromycin B was added to 20 µg/ml immediately before pouring into 10 cm square plates.
Spacing~200 seeds/plate
Average temperature20oC
Average humidity50%
Growth mediumMurashige & Skoog basal salt mixture
Growth medium modifications1x Gamborg's vitamins and 1.5% (w/v) sucrose added and pH adjusted to 5.9. 20 µg/ml hygromycin B added after sterilisation
Growth protocolSeed was surface-sterilised and spread on MS plates supplemented with 1.5% (w/v) sucrose, 0.7% (w/v) agar, 1x Gamborg's vitamins and 20 µg/ml hygromycin B. The plates were sealed, stratified for three days and transferred to an artificial growth room held at a constant 20°C, with a 16h/8h light/dark cycle. Light was provided by fluorescent tubes, generating 85 µmol/m2/s PAR. After ten days, seedlings were transferred from the plates into liquid MS media in a sterile Petri dish, in order to separate the seedlings. Using forceps, individual plants of a similar growth stage (first pair of leaves > 1 mm or Stage 1.02 as defined by Boyes et al., 2001) were selected and placed into 100 ml liquid MS medium in a 250 ml conical flask. Approximately 100 seedlings were transferred, and the flask was sealed with a foam bung. The flask was returned to the growth room and shaken at 76 rpm to provide aeration and adequate mixing. After 48 hours, the seedlings were harvested by filtering through one layer of cheesecloth, and quickly dabbing dry between two pieces of 3MM chromoatography paper. The seedlings were then flash-frozen in liquid nitrogen.
Genetic Variation: transposon gene knock out
Other Information:
AlleleAt2g20570
AlleleAt5g44190

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:0.755854249001
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:
RawQ2.40
NoiseAvg:2.43,Stdev:0.07,Max:2.6,Min:2.3
Central-Avg:5515,Count:9
Corner+Avg:70,Count:32
Corner-Avg:7041,Count:32
BackgroundAvg:56.21,Stdev:0.50,Max:57.3,Min:54.7
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.755854249001
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Waters_1-6_24hr-control_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 ("Waters_1-6_24hr-control_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: Waters_1-6_24hr-control_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Atglk1;Atglk2
Stock Code:
Genetic Background: Col-0
Age: 24hr timepoint
Growth Conditions:
StratificationMS agar plates containing sterilised seed were placed in the dark at 4°C for three days.
SterilisationSeed sterilisation was performed in 15 ml Falcon tubes. Seeds were washed in 70% ethanol for 5 minutes with shaking. The tubes were centrifuged and the ethanol replaced with freshly-prepared 50% sodium hypochlorite solution (BDH; available chlorine %), and the tubes were shaken for 10 minutes. The seeds were pelleted by centrifugation, the bleach solution replaced with sterile pure water, and the tubes shaken vigorously. The seeds were pelleted again and the rinsing was repeated twice more.
OtherTo spread the seed evenly on MS agar plates, the sterilised seeds were resuspended in sterile 0.1% agar and transferred by pipette on to 10 cm square plates. The plates were shaken gently to disperse the seed/agar suspension.
LocationGrowth Room
Growth substrate typeAgar 0.7%
Agar SterilisationAgar was added to a concentration of 0.7% (w/v) agar and the medium was autoclaved at 121 deg. C for 20 mins. The medium was allowed to cool to 50 deg. C. and hygromycin B was added to 20 µg/ml immediately before pouring into 10 cm square plates.
Spacing~200 seeds/plate
Average temperature20oC
Average humidity50%
Growth mediumMurashige & Skoog basal salt mixture
Growth medium modifications1x Gamborg's vitamins and 1.5% (w/v) sucrose added and pH adjusted to 5.9. 20 µg/ml hygromycin B added after sterilisation
Growth protocolSeed was surface-sterilised and spread on MS plates supplemented with 1.5% (w/v) sucrose, 0.7% (w/v) agar, 1x Gamborg's vitamins and 20 µg/ml hygromycin B. The plates were sealed, stratified for three days and transferred to an artificial growth room held at a constant 20°C, with a 16h/8h light/dark cycle. Light was provided by fluorescent tubes, generating 85 µmol/m2/s PAR. After ten days, seedlings were transferred from the plates into liquid MS media in a sterile Petri dish, in order to separate the seedlings. Using forceps, individual plants of a similar growth stage (first pair of leaves > 1 mm or Stage 1.02 as defined by Boyes et al., 2001) were selected and placed into 100 ml liquid MS medium in a 250 ml conical flask. Approximately 100 seedlings were transferred, and the flask was sealed with a foam bung. The flask was returned to the growth room and shaken at 76 rpm to provide aeration and adequate mixing. After 48 hours, the seedlings were harvested by filtering through one layer of cheesecloth, and quickly dabbing dry between two pieces of 3MM chromoatography paper. The seedlings were then flash-frozen in liquid nitrogen.
Genetic Variation: transposon gene knock out
Other Information:
AlleleAt2g20570
AlleleAt5g44190

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:0.683834910393
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:
RawQ2.70
NoiseAvg:2.91,Stdev:0.11,Max:3.2,Min:2.6
Central-Avg:5700,Count:9
Corner+Avg:70,Count:32
Corner-Avg:7058,Count:32
BackgroundAvg:63.82,Stdev:1.18,Max:66.7,Min:60.5
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.683834910393
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Waters_1-7_4hr-induced_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 ("Waters_1-7_4hr-induced_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: Waters_1-7_4hr-induced_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Atglk1;Atglk2
Stock Code:
Genetic Background: Col-0
Age: 4hr timepoint
Growth Conditions:
StratificationMS agar plates containing sterilised seed were placed in the dark at 4°C for three days.
SterilisationSeed sterilisation was performed in 15 ml Falcon tubes. Seeds were washed in 70% ethanol for 5 minutes with shaking. The tubes were centrifuged and the ethanol replaced with freshly-prepared 50% sodium hypochlorite solution (BDH; available chlorine %), and the tubes were shaken for 10 minutes. The seeds were pelleted by centrifugation, the bleach solution replaced with sterile pure water, and the tubes shaken vigorously. The seeds were pelleted again and the rinsing was repeated twice more.
OtherTo spread the seed evenly on MS agar plates, the sterilised seeds were resuspended in sterile 0.1% agar and transferred by pipette on to 10 cm square plates. The plates were shaken gently to disperse the seed/agar suspension.
LocationGrowth Room
Growth substrate typeAgar 0.7%
Agar SterilisationAgar was added to a concentration of 0.7% (w/v) agar and the medium was autoclaved at 121 deg. C for 20 mins. The medium was allowed to cool to 50 deg. C. and hygromycin B was added to 20 µg/ml immediately before pouring into 10 cm square plates.
Spacing~200 seeds/plate
Average temperature20oC
Average humidity50%
Growth mediumMurashige & Skoog basal salt mixture
Growth medium modifications1x Gamborg's vitamins and 1.5% (w/v) sucrose added and pH adjusted to 5.9. 20 µg/ml hygromycin B added after sterilisation
Growth protocolSeed was surface-sterilised and spread on MS plates supplemented with 1.5% (w/v) sucrose, 0.7% (w/v) agar, 1x Gamborg's vitamins and 20 µg/ml hygromycin B. The plates were sealed, stratified for three days and transferred to an artificial growth room held at a constant 20°C, with a 16h/8h light/dark cycle. Light was provided by fluorescent tubes, generating 85 µmol/m2/s PAR. After ten days, seedlings were transferred from the plates into liquid MS media in a sterile Petri dish, in order to separate the seedlings. Using forceps, individual plants of a similar growth stage (first pair of leaves > 1 mm or Stage 1.02 as defined by Boyes et al., 2001) were selected and placed into 100 ml liquid MS medium in a 250 ml conical flask. Approximately 100 seedlings were transferred, and the flask was sealed with a foam bung. The flask was returned to the growth room and shaken at 76 rpm to provide aeration and adequate mixing. After 48 hours, the seedlings were harvested by filtering through one layer of cheesecloth, and quickly dabbing dry between two pieces of 3MM chromoatography paper. The seedlings were then flash-frozen in liquid nitrogen.
Genetic Variation: transposon gene knock out
Other Information:
AlleleAt2g20570
AlleleAt5g44190

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:0.594064354897
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:
RawQ2.48
NoiseAvg:2.64,Stdev:0.08,Max:2.9,Min:2.3
Central-Avg:5574,Count:9
Corner+Avg:78,Count:32
Corner-Avg:7933,Count:32
BackgroundAvg:61.65,Stdev:0.58,Max:62.9,Min:59.5
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.594064354897
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Waters_1-8_4hr-induced_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 ("Waters_1-8_4hr-induced_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: Waters_1-8_4hr-induced_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Atglk1;Atglk2
Stock Code:
Genetic Background: Col-0
Age: 4hr timepoint
Growth Conditions:
StratificationMS agar plates containing sterilised seed were placed in the dark at 4°C for three days.
SterilisationSeed sterilisation was performed in 15 ml Falcon tubes. Seeds were washed in 70% ethanol for 5 minutes with shaking. The tubes were centrifuged and the ethanol replaced with freshly-prepared 50% sodium hypochlorite solution (BDH; available chlorine %), and the tubes were shaken for 10 minutes. The seeds were pelleted by centrifugation, the bleach solution replaced with sterile pure water, and the tubes shaken vigorously. The seeds were pelleted again and the rinsing was repeated twice more.
OtherTo spread the seed evenly on MS agar plates, the sterilised seeds were resuspended in sterile 0.1% agar and transferred by pipette on to 10 cm square plates. The plates were shaken gently to disperse the seed/agar suspension.
LocationGrowth Room
Growth substrate typeAgar 0.7%
Agar SterilisationAgar was added to a concentration of 0.7% (w/v) agar and the medium was autoclaved at 121 deg. C for 20 mins. The medium was allowed to cool to 50 deg. C. and hygromycin B was added to 20 µg/ml immediately before pouring into 10 cm square plates.
Spacing~200 seeds/plate
Average temperature20oC
Average humidity50%
Growth mediumMurashige & Skoog basal salt mixture
Growth medium modifications1x Gamborg's vitamins and 1.5% (w/v) sucrose added and pH adjusted to 5.9. 20 µg/ml hygromycin B added after sterilisation
Growth protocolSeed was surface-sterilised and spread on MS plates supplemented with 1.5% (w/v) sucrose, 0.7% (w/v) agar, 1x Gamborg's vitamins and 20 µg/ml hygromycin B. The plates were sealed, stratified for three days and transferred to an artificial growth room held at a constant 20°C, with a 16h/8h light/dark cycle. Light was provided by fluorescent tubes, generating 85 µmol/m2/s PAR. After ten days, seedlings were transferred from the plates into liquid MS media in a sterile Petri dish, in order to separate the seedlings. Using forceps, individual plants of a similar growth stage (first pair of leaves > 1 mm or Stage 1.02 as defined by Boyes et al., 2001) were selected and placed into 100 ml liquid MS medium in a 250 ml conical flask. Approximately 100 seedlings were transferred, and the flask was sealed with a foam bung. The flask was returned to the growth room and shaken at 76 rpm to provide aeration and adequate mixing. After 48 hours, the seedlings were harvested by filtering through one layer of cheesecloth, and quickly dabbing dry between two pieces of 3MM chromoatography paper. The seedlings were then flash-frozen in liquid nitrogen.
Genetic Variation: transposon gene knock out
Other Information:
AlleleAt2g20570
AlleleAt5g44190

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:0.772210896015
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:
RawQ2.38
NoiseAvg:2.36,Stdev:0.09,Max:2.6,Min:2.1
Central-Avg:5454,Count:9
Corner+Avg:77,Count:32
Corner-Avg:7761,Count:32
BackgroundAvg:53.26,Stdev:0.88,Max:55.1,Min:50.8
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.772210896015
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Waters_1-9_24hr-induced_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 ("Waters_1-9_24hr-induced_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: Waters_1-9_24hr-induced_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Atglk1;Atglk2
Stock Code:
Genetic Background: Col-0
Age: 24hr timepoint
Growth Conditions:
StratificationMS agar plates containing sterilised seed were placed in the dark at 4°C for three days.
SterilisationSeed sterilisation was performed in 15 ml Falcon tubes. Seeds were washed in 70% ethanol for 5 minutes with shaking. The tubes were centrifuged and the ethanol replaced with freshly-prepared 50% sodium hypochlorite solution (BDH; available chlorine %), and the tubes were shaken for 10 minutes. The seeds were pelleted by centrifugation, the bleach solution replaced with sterile pure water, and the tubes shaken vigorously. The seeds were pelleted again and the rinsing was repeated twice more.
OtherTo spread the seed evenly on MS agar plates, the sterilised seeds were resuspended in sterile 0.1% agar and transferred by pipette on to 10 cm square plates. The plates were shaken gently to disperse the seed/agar suspension.
LocationGrowth Room
Growth substrate typeAgar 0.7%
Agar SterilisationAgar was added to a concentration of 0.7% (w/v) agar and the medium was autoclaved at 121 deg. C for 20 mins. The medium was allowed to cool to 50 deg. C. and hygromycin B was added to 20 µg/ml immediately before pouring into 10 cm square plates.
Spacing~200 seeds/plate
Average temperature20oC
Average humidity50%
Growth mediumMurashige & Skoog basal salt mixture
Growth medium modifications1x Gamborg's vitamins and 1.5% (w/v) sucrose added and pH adjusted to 5.9. 20 µg/ml hygromycin B added after sterilisation
Growth protocolSeed was surface-sterilised and spread on MS plates supplemented with 1.5% (w/v) sucrose, 0.7% (w/v) agar, 1x Gamborg's vitamins and 20 µg/ml hygromycin B. The plates were sealed, stratified for three days and transferred to an artificial growth room held at a constant 20°C, with a 16h/8h light/dark cycle. Light was provided by fluorescent tubes, generating 85 µmol/m2/s PAR. After ten days, seedlings were transferred from the plates into liquid MS media in a sterile Petri dish, in order to separate the seedlings. Using forceps, individual plants of a similar growth stage (first pair of leaves > 1 mm or Stage 1.02 as defined by Boyes et al., 2001) were selected and placed into 100 ml liquid MS medium in a 250 ml conical flask. Approximately 100 seedlings were transferred, and the flask was sealed with a foam bung. The flask was returned to the growth room and shaken at 76 rpm to provide aeration and adequate mixing. After 48 hours, the seedlings were harvested by filtering through one layer of cheesecloth, and quickly dabbing dry between two pieces of 3MM chromoatography paper. The seedlings were then flash-frozen in liquid nitrogen.
Genetic Variation: transposon gene knock out
Other Information:
AlleleAt2g20570
AlleleAt5g44190

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:0.688379883766
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:
RawQ2.27
NoiseAvg:2.38,Stdev:0.05,Max:2.5,Min:2.3
Central-Avg:6572,Count:9
Corner+Avg:83,Count:32
Corner-Avg:8549,Count:32
BackgroundAvg:56.88,Stdev:0.46,Max:58.0,Min:55.4
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.688379883766
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015

Slide: Waters_1-10_24hr-induced_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 ("Waters_1-10_24hr-induced_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: Waters_1-10_24hr-induced_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Atglk1;Atglk2
Stock Code:
Genetic Background: Col-0
Age: 24hr timepoint
Growth Conditions:
StratificationMS agar plates containing sterilised seed were placed in the dark at 4°C for three days.
SterilisationSeed sterilisation was performed in 15 ml Falcon tubes. Seeds were washed in 70% ethanol for 5 minutes with shaking. The tubes were centrifuged and the ethanol replaced with freshly-prepared 50% sodium hypochlorite solution (BDH; available chlorine %), and the tubes were shaken for 10 minutes. The seeds were pelleted by centrifugation, the bleach solution replaced with sterile pure water, and the tubes shaken vigorously. The seeds were pelleted again and the rinsing was repeated twice more.
OtherTo spread the seed evenly on MS agar plates, the sterilised seeds were resuspended in sterile 0.1% agar and transferred by pipette on to 10 cm square plates. The plates were shaken gently to disperse the seed/agar suspension.
LocationGrowth Room
Growth substrate typeAgar 0.7%
Agar SterilisationAgar was added to a concentration of 0.7% (w/v) agar and the medium was autoclaved at 121 deg. C for 20 mins. The medium was allowed to cool to 50 deg. C. and hygromycin B was added to 20 µg/ml immediately before pouring into 10 cm square plates.
Spacing~200 seeds/plate
Average temperature20oC
Average humidity50%
Growth mediumMurashige & Skoog basal salt mixture
Growth medium modifications1x Gamborg's vitamins and 1.5% (w/v) sucrose added and pH adjusted to 5.9. 20 µg/ml hygromycin B added after sterilisation
Growth protocolSeed was surface-sterilised and spread on MS plates supplemented with 1.5% (w/v) sucrose, 0.7% (w/v) agar, 1x Gamborg's vitamins and 20 µg/ml hygromycin B. The plates were sealed, stratified for three days and transferred to an artificial growth room held at a constant 20°C, with a 16h/8h light/dark cycle. Light was provided by fluorescent tubes, generating 85 µmol/m2/s PAR. After ten days, seedlings were transferred from the plates into liquid MS media in a sterile Petri dish, in order to separate the seedlings. Using forceps, individual plants of a similar growth stage (first pair of leaves > 1 mm or Stage 1.02 as defined by Boyes et al., 2001) were selected and placed into 100 ml liquid MS medium in a 250 ml conical flask. Approximately 100 seedlings were transferred, and the flask was sealed with a foam bung. The flask was returned to the growth room and shaken at 76 rpm to provide aeration and adequate mixing. After 48 hours, the seedlings were harvested by filtering through one layer of cheesecloth, and quickly dabbing dry between two pieces of 3MM chromoatography paper. The seedlings were then flash-frozen in liquid nitrogen.
Genetic Variation: transposon gene knock out
Other Information:
AlleleAt2g20570
AlleleAt5g44190

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:0.696815669537
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:
RawQ2.28
NoiseAvg:2.46,Stdev:0.09,Max:2.7,Min:2.2
Central-Avg:6795,Count:9
Corner+Avg:103,Count:32
Corner-Avg:9069,Count:32
BackgroundAvg:54.94,Stdev:0.82,Max:56.7,Min:52.8
Gamma1H0.0025
VZ4
Gamma2L0.003
Epsilon0.5
TGT100
Perturbation1.1
Alpha20.06
BF
BG2
Gamma2H0.003
SF0.696815669537
Alpha10.04
SFGeneAll
HZ4
NF1.000000000000
SmoothFactorBG100
Gamma1L0.0025
Tau0.015


Problems? Comments? Suggestions? Contact the Affymetrix Team