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Experiment: Comparison of gonst1 mutant callus with WT

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-704

The aim of this experiment is to compare two independent knockout mutations in the gonst1 gene (At2g13650) with WT. As the plants do not grow on soil, and poorly on agar, we have made callus cell culture lines, from which this RNA is prepared. GONST1 is thought to be a Golgi-localsied GDP-sugar transporter (Baldwin et al., 2001; Handford et al., 2004), although it's precise role in planta is unknown.Baldwin, TC, Handford, MG, Yuseff, I, Orellana, O and P Dupree. (2001) Identification and Characterization of GONST1, a Golgi-Localized GDP- Mannose Transporter in Arabidopsis. The Plant Cell, 13, 2283­-2295Handford MG, Sicilia F, Brandizzi F, Chung JH and P Dupree. (2004) Arabidopsis thaliana expresses multiple Golgi-localised nucleotide-sugar transporters related to GONST1. Molecular and General Genomics, 272, 397-410

About the Experimenter

Name:Dr Jenny Mortimer
Head of Lab Name:Dr Paul Dupree
Lab:
Address:Hopkins Building
Department of Biochemistry
University of Cambridge
Building O, Downing Site
Cambridge
Postcode: CB1 3AW
Country: UK
 
Telephone Number: 01223 333686

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
Number of Slides:6
 
Experimental Parameters:
parametergenetic_modification
Quality Control Measures Taken:
References:
ReferenceBaldwin, TC, Handford, MG, Yuseff, I, Orellana, O and P Dupree. (2001)
ReferenceBaldwin et al., 2001; Handford et al., 2004
ReferenceThe Plant Cell, 13, 2283­-2295 Handford MG, Sicilia F, Brandizzi F, Chung JH and P Dupree. (2004)
ReferenceMolecular and General Genomics, 272, 397-410
 
Other Information:

Slides in this Experiment

Hybridisation Set: Mortimer: Comparison of gonst1 mutant callus with WT

Slide: Mortimer_704-1_Col0-callus_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_704-1_Col0-callus_Rep1_ATH1") was made from "" 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_704-1_Col0-callus_Rep1_ATH1
Organism:
Alias: Col-0
Stock Code: N1092
Genetic Background: Col-0 (Columbia, N60000)
Growth Conditions:
StratificationSeedlings for callus: after sterilisation and sowing onto standard MS plates, plates were stored at 4'C, for 2 days in the dark to stratify.
SterilisationSeeds were suspended in 70% (v/v) ethanol (200 ul), and then quickly washed with sterile water (1 ml) 4-5 times. Following a 5 minute incubation in Milton's bleach (200 ul), the seeds were washed a futher 4-5 times.
ProtocolCallus induction and maintenance After straification, plants were moved to the light (16hL:8hD; 25'C) for 10-14 d. Roots were removed, sliced, and placed individually on callus induction medium plates (see below for recipe) in low light, 12h days, at 25 'C. After 3-4 weeks, the small calli which have formed were tranferred to fresh plates, leaving root material behind. Calli were sub-cultured at least twice, and then transferred to 50 ml Gamborg's medium (110 rpm, 25'C, subdued light). Medium was refreshed weekly, and cells were harvested after the second medium change. Callus induction medium and culture medium 3.86g Gamborg's B5 Medium inc. vitamins (Sigma G5893) 20g glucose 0.5g MES 4.5mls 2,4-D stock (final 0.5mg/l) 50microl. kinetin (1mg/ml stock) Adjust to pH 5.7 with 1M KOH and make up to 1 litre. Store at -20C or use immediately. For plates: add 8g/l agar. For liquid culture: 50ml in 250ml flask or 200ml in 1l flask.
ReferenceWee, G.-T., Sherrier, D. J., Prime, T. A., Dupree, P., Plant Cell 1998, 10, 1759-1768.
Hydroponics Gamborgs medium
Substrate Sterilising ProcedureAutoclaved
Temperature25 °C average, 25 °C day, 25 °C night
Humidity60 % average, 60 % day, 60 % night
MediumGamborg's B-5 basal salt mixture. Frequency: Weekly
Lighting(Source: Day length:720, Night Length:720. Wavelength: White)No constant light
Tissue: Callus cells
Additional Organism Information:
Sample DescriptionCallus cells
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:0.435582
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-ratio1.086434
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.952304
Spike_AFFX-r2-Bs-dap_5_signal60.419106
NoiseAvg:3.02,Std:0.09,Min:2.8,Max:3.2
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal17.348976
#P14139
Spike_AFFX-r2-Bs-phe_M_signal44.407631
Corner-Avg:15295,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal92.773048
Spike_AFFX-r2-Ec-bioB_3_signal55.231144
Spike_AFFX-r2-Bs-lys_M_signal28.267471
Spike_AFFX-r2-P1-cre_3_signal3561.383057
Spike_AFFX-r2-Bs-lys_3-5-ratio1.674845
Spike_AFFX-r2-Bs-dap_M_signal216.828354
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio13.646844
Spike_AFFX-r2-Ec-bioB_avg-signal68.667191
Spike_AFFX-r2-Bs-thr_avg-signal109.462868
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal693.097107
Spike_AFFX-r2-Bs-phe_5_signal35.702885
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal973.643127
RawQ2.631680
Spike_AFFX-r2-Bs-lys_5_signal20.696369
Signal(A)3.224803
%A36.405087
Signal(All)141.747437
Corner+Avg:130,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal236.116562
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal98.294373
Spike_AFFX-r2-Ec-bioD_avg-signal833.370117
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P61.985970
Spike_AFFX-r2-Bs-lys_avg-signal27.875681
Spike_AFFX-r2-P1-cre_avg-signal3419.714844
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.608943
Spike_AFFX-r2-Bs-thr_3_signal236.758774
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-signal59.468292
Spike_AFFX-r2-Bs-dap_3_signal433.491364
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.404772
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal211.123337
#M367
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal34.663208
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.118382
Spike_AFFX-r2-Bs-thr_M_signal74.280876
Signal(P)226.447876
Spike_AFFX-r2-Bs-phe_3-5-ratio2.753121
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:13908,Count:9
Spike_AFFX-r2-P1-cre_5_signal3278.046631
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8304
Signal(M)12.898597
BackgroundAvg:65.96,Std:0.86,Min:62.1,Max:67.7
Spike_AFFX-r2-Ec-bioC_avg-signal223.619949
Spike_AFFX-r2-Bs-dap_avg-signal236.912949
Spike_AFFX-r2-Bs-dap_3-5-ratio7.174740
Spike_AFFX-r2-Ec-bioB_5_signal57.997372
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.435582
NF1.000000
HZ4
Tau0.015000

Slide: Mortimer_704-2_Col0-callus_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_704-2_Col0-callus_Rep2_ATH1") was made from "" 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_704-2_Col0-callus_Rep2_ATH1
Organism:
Alias: Col-0
Stock Code: N1092
Genetic Background: Col-0 (Columbia, N60000)
Growth Conditions:
StratificationSeedlings for callus: after sterilisation and sowing onto standard MS plates, plates were stored at 4'C, for 2 days in the dark to stratify.
SterilisationSeeds were suspended in 70% (v/v) ethanol (200 ul), and then quickly washed with sterile water (1 ml) 4-5 times. Following a 5 minute incubation in Milton's bleach (200 ul), the seeds were washed a futher 4-5 times.
ProtocolCallus induction and maintenance After straification, plants were moved to the light (16hL:8hD; 25'C) for 10-14 d. Roots were removed, sliced, and placed individually on callus induction medium plates (see below for recipe) in low light, 12h days, at 25 'C. After 3-4 weeks, the small calli which have formed were tranferred to fresh plates, leaving root material behind. Calli were sub-cultured at least twice, and then transferred to 50 ml Gamborg's medium (110 rpm, 25'C, subdued light). Medium was refreshed weekly, and cells were harvested after the second medium change. Callus induction medium and culture medium 3.86g Gamborg's B5 Medium inc. vitamins (Sigma G5893) 20g glucose 0.5g MES 4.5mls 2,4-D stock (final 0.5mg/l) 50microl. kinetin (1mg/ml stock) Adjust to pH 5.7 with 1M KOH and make up to 1 litre. Store at -20C or use immediately. For plates: add 8g/l agar. For liquid culture: 50ml in 250ml flask or 200ml in 1l flask.
ReferenceWee, G.-T., Sherrier, D. J., Prime, T. A., Dupree, P., Plant Cell 1998, 10, 1759-1768.
Hydroponics Gamborgs medium
Substrate Sterilising ProcedureAutoclaved
Temperature25 °C average, 25 °C day, 25 °C night
Humidity60 % average, 60 % day, 60 % night
MediumGamborg's B-5 basal salt mixture. Frequency: Weekly
Lighting(Source: Day length:720, Night Length:720. Wavelength: White)No constant light
Tissue: Callus cells
Additional Organism Information:
Sample DescriptionCallus cells
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:0.612041
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-ratio1.121121
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.885195
Spike_AFFX-r2-Bs-dap_5_signal85.469887
NoiseAvg:3.40,Std:0.12,Min:2.9,Max:3.7
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal18.260332
#P13330
Spike_AFFX-r2-Bs-phe_M_signal65.273911
Corner-Avg:12077,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal70.334145
Spike_AFFX-r2-Ec-bioB_3_signal31.065620
Spike_AFFX-r2-Bs-lys_M_signal30.135365
Spike_AFFX-r2-P1-cre_3_signal2630.772217
Spike_AFFX-r2-Bs-lys_3-5-ratio1.692782
Spike_AFFX-r2-Bs-dap_M_signal351.382477
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio17.670750
Spike_AFFX-r2-Ec-bioB_avg-signal45.498138
Spike_AFFX-r2-Bs-thr_avg-signal152.997955
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal398.075897
Spike_AFFX-r2-Bs-phe_5_signal65.367241
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal551.898743
RawQ3.029276
Spike_AFFX-r2-Bs-lys_5_signal27.760454
Signal(A)5.179662
%A39.592285
Signal(All)143.737762
Corner+Avg:104,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal162.004944
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal116.245331
Spike_AFFX-r2-Ec-bioD_avg-signal474.987305
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P58.439281
Spike_AFFX-r2-Bs-lys_avg-signal34.962742
Spike_AFFX-r2-P1-cre_avg-signal2488.663818
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.968435
Spike_AFFX-r2-Bs-thr_3_signal322.673767
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-signal82.295494
Spike_AFFX-r2-Bs-dap_3_signal545.710876
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.386416
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal110.085007
#M449
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal46.992409
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.471635
Spike_AFFX-r2-Bs-thr_M_signal118.059753
Signal(P)241.819092
Spike_AFFX-r2-Bs-phe_3-5-ratio1.778342
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:10357,Count:9
Spike_AFFX-r2-P1-cre_5_signal2346.555420
Spike_AFFX-r2-Bs-phe_M_detectionP
#A9031
Signal(M)18.780500
BackgroundAvg:75.02,Std:1.23,Min:71.9,Max:79.9
Spike_AFFX-r2-Ec-bioC_avg-signal136.044983
Spike_AFFX-r2-Bs-dap_avg-signal327.521088
Spike_AFFX-r2-Bs-dap_3-5-ratio6.384832
Spike_AFFX-r2-Ec-bioB_5_signal35.094643
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.612041
NF1.000000
HZ4
Tau0.015000

Slide: Mortimer_704-3_gonst1-2-callus_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 ("Mortimer_704-3_gonst1-2-callus_Rep1_ATH1") was made from "" 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_704-3_gonst1-2-callus_Rep1_ATH1
Organism:
Alias: SALK_043593
Stock Code: N543593
Genetic Background: Col-0 (Columbia, N60000)
Growth Conditions:
StratificationFor production of callus: Following sterilisation, seeds were plated onto MS agar, and stratified at 4'C, in the dark for 2 days.
SterilisationSeeds were washed in 70 % (v/v) ethanol, and then rinsed 4-5 times in sterile distilled water. The seeds were then incubated in Milton's bleach (200 ul) for 5 minutes, followed by 4-5 washed in sterile distilled water.
ProtocolFollowing growth in the light (16hL:8hD; 25'C) for 10-14 days, the roots were excised and sliced up using a sterile razor blade. These fragments were transferred to callus induction medium (see below for recipe) and cultured for 3-4 weeks under subdued light (12hL:12hD; 25'C). The calli were transferred to fresh plates, leaving any root tissue behind. Calli that had been subcultured at least twice were transferred to liquid culture (50 ml Gamborgs medium; 110 rpm, subdued light; 12hL:12hD). Liquid medium was refreshed once a week, and cells harvested after 2 weeks. Callus induction medium and culture medium 3.86g Gamborg's B5 Medium inc. vitamins (Sigma G5893) 20g glucose 0.5g MES 4.5mls 2,4-D stock (final 0.5mg/l) 50microl. kinetin (1mg/ml stock) Adjust to pH 5.7 with 1M KOH and make up to 1 litre. Store at -20C or use immediately. For plates: add 8g/l agar. For liquid culture: 50ml in 250ml flask.
ReferenceWee, G.-T., Sherrier, D. J., Prime, T. A., Dupree, P., Plant Cell 1998, 10, 1759-1768.
Hydroponics Gamborgs medium
Substrate Sterilising ProcedureAutoclaved
Temperature25 °C average, 25 °C day, 25 °C night
Humidity60 % average, 60 % day, 60 % night
MediumGamborg's B-5 basal salt mixture. Frequency: Weekly
Lighting(Source: Day length:720, Night Length:720. Wavelength: White)No constant light
Tissue: whole plant
Additional Organism Information:
Sample DescriptionCallus suspension cell culture
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:0.603146
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-ratio1.056861
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.890013
Spike_AFFX-r2-Bs-dap_5_signal109.933769
NoiseAvg:2.73,Std:0.07,Min:2.5,Max:2.9
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal34.302643
#P13824
Spike_AFFX-r2-Bs-phe_M_signal73.081093
Corner-Avg:14076,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal92.637993
Spike_AFFX-r2-Ec-bioB_3_signal62.131744
Spike_AFFX-r2-Bs-lys_M_signal38.454647
Spike_AFFX-r2-P1-cre_3_signal3673.753174
Spike_AFFX-r2-Bs-lys_3-5-ratio1.691850
Spike_AFFX-r2-Bs-dap_M_signal345.148102
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio11.186305
Spike_AFFX-r2-Ec-bioB_avg-signal74.859886
Spike_AFFX-r2-Bs-thr_avg-signal176.351730
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal618.316223
Spike_AFFX-r2-Bs-phe_5_signal69.146469
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal932.426208
RawQ2.456216
Spike_AFFX-r2-Bs-lys_5_signal37.505383
Signal(A)3.786865
%A37.623848
Signal(All)141.956604
Corner+Avg:108,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal235.501328
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal145.822327
Spike_AFFX-r2-Ec-bioD_avg-signal775.371216
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P60.605000
Spike_AFFX-r2-Bs-lys_avg-signal46.471172
Spike_AFFX-r2-P1-cre_avg-signal3574.926270
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.771153
Spike_AFFX-r2-Bs-thr_3_signal383.719818
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-signal96.016624
Spike_AFFX-r2-Bs-dap_3_signal631.787537
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.508009
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal213.403717
#M404
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal63.453484
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.103548
Spike_AFFX-r2-Bs-thr_M_signal111.032730
Signal(P)231.458954
Spike_AFFX-r2-Bs-phe_3-5-ratio2.108891
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:11835,Count:9
Spike_AFFX-r2-P1-cre_5_signal3476.099365
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8582
Signal(M)14.461779
BackgroundAvg:60.34,Std:0.86,Min:58.2,Max:62.3
Spike_AFFX-r2-Ec-bioC_avg-signal224.452515
Spike_AFFX-r2-Bs-dap_avg-signal362.289795
Spike_AFFX-r2-Bs-dap_3-5-ratio5.746984
Spike_AFFX-r2-Ec-bioB_5_signal69.809914
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.603146
NF1.000000
HZ4
Tau0.015000

Slide: Mortimer_704-4_gonst1-2-callus_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_704-4_gonst1-2-callus_Rep2_ATH1") was made from "" 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_704-4_gonst1-2-callus_Rep2_ATH1
Organism:
Alias: SALK_043593
Stock Code: N543593
Genetic Background: Col-0 (Columbia, N60000)
Growth Conditions:
StratificationFor production of callus: Following sterilisation, seeds were plated onto MS agar, and stratified at 4'C, in the dark for 2 days.
SterilisationSeeds were washed in 70 % (v/v) ethanol, and then rinsed 4-5 times in sterile distilled water. The seeds were then incubated in Milton's bleach (200 ul) for 5 minutes, followed by 4-5 washed in sterile distilled water.
ProtocolFollowing growth in the light (16hL:8hD; 25'C) for 10-14 days, the roots were excised and sliced up using a sterile razor blade. These fragments were transferred to callus induction medium (see below for recipe) and cultured for 3-4 weeks under subdued light (12hL:12hD; 25'C). The calli were transferred to fresh plates, leaving any root tissue behind. Calli that had been subcultured at least twice were transferred to liquid culture (50 ml Gamborgs medium; 110 rpm, subdued light; 12hL:12hD). Liquid medium was refreshed once a week, and cells harvested after 2 weeks. Callus induction medium and culture medium 3.86g Gamborg's B5 Medium inc. vitamins (Sigma G5893) 20g glucose 0.5g MES 4.5mls 2,4-D stock (final 0.5mg/l) 50microl. kinetin (1mg/ml stock) Adjust to pH 5.7 with 1M KOH and make up to 1 litre. Store at -20C or use immediately. For plates: add 8g/l agar. For liquid culture: 50ml in 250ml flask.
ReferenceWee, G.-T., Sherrier, D. J., Prime, T. A., Dupree, P., Plant Cell 1998, 10, 1759-1768.
Hydroponics Gamborgs medium
Substrate Sterilising ProcedureAutoclaved
Temperature25 °C average, 25 °C day, 25 °C night
Humidity60 % average, 60 % day, 60 % night
MediumGamborg's B-5 basal salt mixture. Frequency: Weekly
Lighting(Source: Day length:720, Night Length:720. Wavelength: White)No constant light
Tissue: whole plant
Additional Organism Information:
Sample DescriptionCallus suspension cell culture
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:0.879566
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-ratio1.103544
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.104561
Spike_AFFX-r2-Bs-dap_5_signal72.267929
NoiseAvg:2.23,Std:0.08,Min:2.1,Max:2.5
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal20.390640
#P13872
Spike_AFFX-r2-Bs-phe_M_signal65.055878
Corner-Avg:10766,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal129.474030
Spike_AFFX-r2-Ec-bioB_3_signal61.533150
Spike_AFFX-r2-Bs-lys_M_signal37.614113
Spike_AFFX-r2-P1-cre_3_signal5068.616211
Spike_AFFX-r2-Bs-lys_3-5-ratio2.147963
Spike_AFFX-r2-Bs-dap_M_signal308.148315
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio22.708309
Spike_AFFX-r2-Ec-bioB_avg-signal82.238464
Spike_AFFX-r2-Bs-thr_avg-signal198.908798
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal802.804077
Spike_AFFX-r2-Bs-phe_5_signal53.066044
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1211.234985
RawQ2.112390
Spike_AFFX-r2-Bs-lys_5_signal32.085918
Signal(A)4.373161
%A37.417801
Signal(All)147.214005
Corner+Avg:88,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal298.779999
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal137.559570
Spike_AFFX-r2-Ec-bioD_avg-signal1007.019531
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P60.815434
Spike_AFFX-r2-Bs-lys_avg-signal46.206467
Spike_AFFX-r2-P1-cre_avg-signal4830.825684
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.766769
Spike_AFFX-r2-Bs-thr_3_signal463.036957
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-signal85.227165
Spike_AFFX-r2-Bs-dap_3_signal590.502930
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.508755
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal260.721619
#M403
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal68.919373
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.145973
Spike_AFFX-r2-Bs-thr_M_signal113.298805
Signal(P)238.888901
Spike_AFFX-r2-Bs-phe_3-5-ratio2.592233
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:8897,Count:9
Spike_AFFX-r2-P1-cre_5_signal4593.035156
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8535
Signal(M)16.772930
BackgroundAvg:50.57,Std:1.08,Min:48.6,Max:53.3
Spike_AFFX-r2-Ec-bioC_avg-signal279.750793
Spike_AFFX-r2-Bs-dap_avg-signal323.639740
Spike_AFFX-r2-Bs-dap_3-5-ratio8.171023
Spike_AFFX-r2-Ec-bioB_5_signal55.708221
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.879566
NF1.000000
HZ4
Tau0.015000

Slide: Mortimer_704-5_gonst1-3-callus_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 ("Mortimer_704-5_gonst1-3-callus_Rep1_ATH1") was made from "" 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_704-5_gonst1-3-callus_Rep1_ATH1
Organism:
Alias: Col-0
Stock Code:
Genetic Background: Col-0 (Columbia, N60000)
Growth Conditions:
StratificationFor production of callus: Following sterilisation, seeds were plated onto MS agar, and stratified at 4'C, in the dark for 2 days.
SterilisationSeeds were washed in 70 % (v/v) ethanol, and then rinsed 4-5 times in sterile distilled water. The seeds were then incubated in Milton's bleach (200 ul) for 5 minutes, followed by 4-5 washed in sterile distilled water.
ProtocolFollowing growth in the light (16hL:8hD; 25'C) for 10-14 days, the roots were excised and sliced up using a sterile razor blade. These fragments were transferred to callus induction medium (see below for recipe) and cultured for 3-4 weeks under subdued light (12hL:12hD; 25'C). The calli were transferred to fresh plates, leaving any root tissue behind. Calli that had been subcultured at least twice were transferred to liquid culture (50 ml Gamborgs medium; 110 rpm, subdued light; 12hL:12hD). Liquid medium was refreshed once a week, and cells harvested after 2 weeks. Callus induction medium and culture medium 3.86g Gamborg's B5 Medium inc. vitamins (Sigma G5893) 20g glucose 0.5g MES 4.5mls 2,4-D stock (final 0.5mg/l) 50microl. kinetin (1mg/ml stock) Adjust to pH 5.7 with 1M KOH and make up to 1 litre. Store at -20C or use immediately. For plates: add 8g/l agar. For liquid culture: 50ml in 250ml flask.
ReferenceWee, G.-T., Sherrier, D. J., Prime, T. A., Dupree, P., Plant Cell 1998, 10, 1759-1768.
Hydroponics Gamborgs medium
Substrate Sterilising ProcedureAutoclaved
Temperature25 °C average, 25 °C day, 25 °C night
Humidity60 % average, 60 % day, 60 % night
MediumGamborg's B-5 basal salt mixture. Frequency: Weekly
Lighting(Source: Day length:720, Night Length:720. Wavelength: White)No constant light
Tissue: whole plant
Additional Organism Information:
Sample DescriptionCallus suspension cell culture
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:0.582846
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-ratio1.039876
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.942090
Spike_AFFX-r2-Bs-dap_5_signal111.476730
NoiseAvg:2.52,Std:0.09,Min:2.2,Max:2.8
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal21.750349
#P14061
Spike_AFFX-r2-Bs-phe_M_signal67.502823
Corner-Avg:13082,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal90.420311
Spike_AFFX-r2-Ec-bioB_3_signal55.787193
Spike_AFFX-r2-Bs-lys_M_signal32.643826
Spike_AFFX-r2-P1-cre_3_signal3902.844971
Spike_AFFX-r2-Bs-lys_3-5-ratio1.585887
Spike_AFFX-r2-Bs-dap_M_signal347.194244
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio13.451933
Spike_AFFX-r2-Ec-bioB_avg-signal68.474640
Spike_AFFX-r2-Bs-thr_avg-signal142.131378
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal690.119507
Spike_AFFX-r2-Bs-phe_5_signal72.357986
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal991.952698
RawQ2.327219
Spike_AFFX-r2-Bs-lys_5_signal41.504990
Signal(A)3.268035
%A36.900482
Signal(All)143.295837
Corner+Avg:103,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal222.991119
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal132.260956
Spike_AFFX-r2-Ec-bioD_avg-signal841.036133
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P61.644016
Spike_AFFX-r2-Bs-lys_avg-signal46.657013
Spike_AFFX-r2-P1-cre_avg-signal3828.014893
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.455502
Spike_AFFX-r2-Bs-thr_3_signal292.584229
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-signal90.707253
Spike_AFFX-r2-Bs-dap_3_signal671.172302
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.437364
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal203.849854
#M332
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal65.822220
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.093899
Spike_AFFX-r2-Bs-thr_M_signal112.059563
Signal(P)230.184235
Spike_AFFX-r2-Bs-phe_3-5-ratio1.827870
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:12016,Count:9
Spike_AFFX-r2-P1-cre_5_signal3753.184814
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8417
Signal(M)13.405457
BackgroundAvg:57.35,Std:0.64,Min:54.8,Max:58.5
Spike_AFFX-r2-Ec-bioC_avg-signal213.420486
Spike_AFFX-r2-Bs-dap_avg-signal376.614410
Spike_AFFX-r2-Bs-dap_3-5-ratio6.020739
Spike_AFFX-r2-Ec-bioB_5_signal59.216415
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.582846
NF1.000000
HZ4
Tau0.015000

Slide: Mortimer_704-6_gonst1-3-callus_Rep2_ATH1

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

Summary Information

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

  • Extract 1 ("Mortimer_704-6_gonst1-3-callus_Rep2_ATH1") was made from "" 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_704-6_gonst1-3-callus_Rep2_ATH1
Organism:
Alias: Col-0
Stock Code:
Genetic Background: Col-0 (Columbia, N60000)
Growth Conditions:
StratificationFor production of callus: Following sterilisation, seeds were plated onto MS agar, and stratified at 4'C, in the dark for 2 days.
SterilisationSeeds were washed in 70 % (v/v) ethanol, and then rinsed 4-5 times in sterile distilled water. The seeds were then incubated in Milton's bleach (200 ul) for 5 minutes, followed by 4-5 washed in sterile distilled water.
ProtocolFollowing growth in the light (16hL:8hD; 25'C) for 10-14 days, the roots were excised and sliced up using a sterile razor blade. These fragments were transferred to callus induction medium (see below for recipe) and cultured for 3-4 weeks under subdued light (12hL:12hD; 25'C). The calli were transferred to fresh plates, leaving any root tissue behind. Calli that had been subcultured at least twice were transferred to liquid culture (50 ml Gamborgs medium; 110 rpm, subdued light; 12hL:12hD). Liquid medium was refreshed once a week, and cells harvested after 2 weeks. Callus induction medium and culture medium 3.86g Gamborg's B5 Medium inc. vitamins (Sigma G5893) 20g glucose 0.5g MES 4.5mls 2,4-D stock (final 0.5mg/l) 50microl. kinetin (1mg/ml stock) Adjust to pH 5.7 with 1M KOH and make up to 1 litre. Store at -20C or use immediately. For plates: add 8g/l agar. For liquid culture: 50ml in 250ml flask.
ReferenceWee, G.-T., Sherrier, D. J., Prime, T. A., Dupree, P., Plant Cell 1998, 10, 1759-1768.
Hydroponics Gamborgs medium
Substrate Sterilising ProcedureAutoclaved
Temperature25 °C average, 25 °C day, 25 °C night
Humidity60 % average, 60 % day, 60 % night
MediumGamborg's B-5 basal salt mixture. Frequency: Weekly
Lighting(Source: Day length:720, Night Length:720. Wavelength: White)No constant light
Tissue: whole plant
Additional Organism Information:
Sample DescriptionCallus suspension cell culture
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:0.467622
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-ratio1.017763
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.979504
Spike_AFFX-r2-Bs-dap_5_signal92.348549
NoiseAvg:2.66,Std:0.09,Min:2.4,Max:3.0
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal21.576283
#P14269
Spike_AFFX-r2-Bs-phe_M_signal71.248062
Corner-Avg:13504,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal79.081413
Spike_AFFX-r2-Ec-bioB_3_signal55.466293
Spike_AFFX-r2-Bs-lys_M_signal41.979107
Spike_AFFX-r2-P1-cre_3_signal3367.701904
Spike_AFFX-r2-Bs-lys_3-5-ratio2.128779
Spike_AFFX-r2-Bs-dap_M_signal371.943542
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio17.789942
Spike_AFFX-r2-Ec-bioB_avg-signal63.724880
Spike_AFFX-r2-Bs-thr_avg-signal176.451523
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal659.202332
Spike_AFFX-r2-Bs-phe_5_signal72.478577
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal962.459473
RawQ2.469011
Spike_AFFX-r2-Bs-lys_5_signal30.478395
Signal(A)2.863786
%A36.124508
Signal(All)139.917023
Corner+Avg:104,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal194.486786
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal172.823929
Spike_AFFX-r2-Ec-bioD_avg-signal810.830933
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P62.555897
Spike_AFFX-r2-Bs-lys_avg-signal45.779755
Spike_AFFX-r2-P1-cre_avg-signal3338.314453
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.319597
Spike_AFFX-r2-Bs-thr_3_signal383.840820
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-signal105.516853
Spike_AFFX-r2-Bs-dap_3_signal684.732056
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.460037
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal178.748352
#M301
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal64.881760
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.088048
Spike_AFFX-r2-Bs-thr_M_signal123.937477
Signal(P)221.757858
Spike_AFFX-r2-Bs-phe_3-5-ratio2.384483
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:10987,Count:9
Spike_AFFX-r2-P1-cre_5_signal3308.926758
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8240
Signal(M)12.115850
BackgroundAvg:60.14,Std:0.79,Min:58.1,Max:62.5
Spike_AFFX-r2-Ec-bioC_avg-signal186.617569
Spike_AFFX-r2-Bs-dap_avg-signal383.008026
Spike_AFFX-r2-Bs-dap_3-5-ratio7.414649
Spike_AFFX-r2-Ec-bioB_5_signal56.626938
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.467622
NF1.000000
HZ4
Tau0.015000


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