10 protocols
AccessionNameType
P-MTAB-33692
array scanning protocol
Scanning parameters were adjusted to Cy5 PMT=72, Cy3 PMT=82.

2 separate images are acquired for each dye for the whole array.

Intensities of the fluorescent signals were quantified using AIDA (raytest GmbH, Straubenhardt, Germany).

The red channel (Cy5) was linked to index 1. The green channel (Cy3) was linked to index 2. Default spot size was set to 0.16 mm and allowed to be adjusted to a minimum of 0.10 mm. Background quantification was set to "Median, local dot ring, 0.03 mm, no dot inflation". Quality controls were set to "Rate saturated pixel when saturated area > 10%, Rate dot homogeneity: Full diameter, Deviation > 25%, Signal > 5*Bkg". No normalization was applied.


(Parameters: Scanning hardware = OTHER: Fuji FLA-3000, Scanning software = Scanning software)
P-MTAB-33700
nucleic acid hybridization to array protocol
Hybridisation Materials 1. spotted microarrays on Type 7* mirror slides (Amersham Pharmacia Biotech, RPK2331) 2. Poly-dT35, 1 µg/µL (Metabion) 3. Microarray hybridization buffer (Amersham Pharmacia Biotech, RPK0325) 4. Formamide 5. Glass cover slips, (24×50 to 24×60 mm) 6. 20× SSPE 7. 10% SDS 8. distilled water Protocol Hybridisation Make up the following mixture: 2 µg (2 µL) Poly-dT35 ¼ Vol. (15 µL) Microarray hybridization buffer 50,00% (30 µL) Formamide to 60 µL (13 µL) water dissolve the 1 pmol of Cy3 labeled oligonucleotide and Cy5-labeled sample at a quantity of 40 pmoles of incorporated dye in the mixture incubate for 3 min 96 °C chill on ice and leave it there until hybridisation incubate the microarray in 2× SSPE + 0,2% SDS for 30 min, RT dip the microarray a few times in distilled water dry the microarray by air stream pipet the complete sample onto the microarray cover the microarray carefully with a coverslip; avoid the formation of bubbles! insert the microarray into an incubation chamber incubate over night at 42 °C Washing Preparations: pre-warm a staining chamber with 1× SSC + 0,2% SDS to 55 °C pre-warm two staining chambers with 0,1× SSC + 0,2% SDS to 55 °C pre-warm a staining chamber with 0,1× SSC to 37 °C keep one staining chamber with distilled water at RT Protocol: remove the cover slip from the microarray by carefully dipping in 1× SSC + 0,2% SDS wash for 10 min, 55 ° C in 1× SSC + 0,2% SDS wash for 10 min, 55 ° C in 0,1× SSC + 0,2% SDS wash for 10 min, 55 ° C in 0,1× SSC + 0,2% SDS wash for 1 min, 37 ° C in 0,1× SSC dip five times in distilled water dry by air stream
(Parameters: Chamber type = OTHER: SCIENION hybridization chamber, Quantity of label target used = 50, Mass unit = Micro gram, time = 16, Tiny time unit = hours, Volume = 60, Volume unit = Micro litre, temperature = 42)
P-MTAB-33699
nucleic acid labeling protocol
A Cy3 labeled oligonucleotide against the minuteary primers used for GST amplification was distributed by Paul van Hummelen to the partners of the CAGE project.
(Parameters: Mass unit = Micro gram, Label used = Cy3, Amplification = none)
P-MTAB-33698
growth protocol
This is a dummy protocol for labeled oligos.
(Parameters: time unit = minutes, temperature unit = C)
P-MTAB-33696
treatment protocol
This is a dummy protocol for the labeled oligonucleotide.
P-MTAB-33697
nucleic acid extraction protocol
This is a dummy protocol for labeled oligonucleotides.
(Parameters: Extracted product = OTHER: none, Amplification = none)
P-MTAB-33695
nucleic acid labeling protocol
Materials 1. RNase Out, 40 U/µL (Invitrogen 10777-019) 2. HT7T-Primer, HPLC-purified, 200 ng/µL. Sequence: 5'-GGC. CAG.TGA.ATT.GTA.ATA.CGA.CTC.ACT.ATA.GGG.AGG.CGG.TTT.TTT.TTT.TTT.TTT.TTT. TTT.TTT-3' 3. 1,7 M Trehalose (Sigma T-5251) 4. SuperScript II RT RNase H – reverse transcriptase, 200 U/µL (Invitrogen, 10.000 U, 18064-014) package contains: SuperScript II RT, 5× first strand buffer, 0,1M DTT 5. 10 mM dNTP-Mix (Amersham Pharmacia Biotech, 100 mM, 27-2035-01) 6. 5× minute strand buffer: 100 mM Tris-HCl, pH 6,9 450 mM KCl 23 mM MgCl2 0,75 mM • -NAD + (Sigma, N-1636) 50 mM (NH4)2SO4 7. E. coli DNA-Ligase, 10 U/µL (Invitrogen, 100 U, 18052-019) 8. E. coli DNA-Polymerase I, 10 U/µL (Invitrogen, 1000 U, 18010-025) 9. E. coli RNase H, 2 U/µL (Invitrogen, 120 U, 18021-017) 10. DEPC-Wasser (Invitrogen, 10977-015) 11. Ethanol absolute 12. 3 M Sodium acetate, pH5,0 with glacial acetic acid 13. MegaScript T7 transcription kit (Ambion, 1334) 14. QiaQuick PCR purification kit (Qiagen, 28106) 15. RNeasy mini kit (Qiagen, 74106) Protocol First strand synthesis Preparations: dissolve Trehalose at > 42 ° C completely under frequent mixing pre-warm a heating block to 75 °C thaw 5× first strand buffer, DTT and dNTP-Mix and place on ice Make up the following reaction: 5 µg Total RNA preparation 40U (1 µL) RNase Out adjust volume with RNase-free water to 4 µL, or dry out in a SpeedVac 200 ng (1 µL) HT7T-Primer 10,2 micromolarol (6 µL) 1,7 M Trehalose incubate for 5 min, 75 ° C then chill on ice and spin down 1/5 Vol. (4 µL) 5× first strand buffer 200 nmol (2 µL) 0,1 M DTT# 10 nmol (1 µL) 10 mM dNTP-Mix 1,7 micromolarol (1 µL) 1,7 M Trehalose 200 U (1 µL) SuperScript II RT transfer the mixture into 0.2 mL tubes and incubate as followed: 1. 5 min 37 °C 2. 5 min 45 °C 3. 2 min 60 °C 4. 2 min 55 °C 5. repeat 9 more times from step 3 6. 10 min 65 °C 7. keep at 4 °C minute strand synthesis: Make up the following reaction: complete first strand product (20 µL) 1/5 Vol. (33,4 µL) 5× minute strand buffer 34 nmol (3,4 µL) 10 mM dNTP-Mix 10 U (1 µL) E. coli DNA-Ligase 40 U (4 µL) E. coli DNA-Polymerase I 2 U (1 µL) E. coli RNase H up to 166,6 µL (103,8 µL) water incubate for 3 h, 16 °C incubate for 30 min 65 °C keep at 4 °C or on ice Purification with QiaQuick PCR purification kit 1. add 7 µL 3 M Sodium acetate to the minute strand product 2. add 5 Vol. Buffer PB (usually 868 µL) 3. mix well and incubate for 2 – 3 min on ice 4. apply half of the mixture onto a QiaQuick column 5. centrifuge at 9000 rpm, 15 s, RT 6. discard the filtrate, place the column back into the receiver tube 7. apply the remaining mixture onto the column 8. centrifuge at 9000 rpm, 15 s, RT 9. discard the filtrate, place the column back into the receiver tube 10. add 500 µL Buffer PE onto the column 11. centrifuge at 9000 rpm, 15 s, RT 12. discard the filtrate, place the column back into the receiver tube 13. add another 500 µL Buffer PE onto the column 14. centrifuge at 9000 rpm, 15 s, RT 15. discard the filtrate, place the column back into the receiver tube 16. centrifuge at maximum speed, 1 min, RT 17. transfer the column to a fresh 1.5 mL tube 18. add 45 µL 1/5 EB onto the column 19. incubate for 1 min, RT 20. centrifuge at 9000 rpm, 15 s, RT 21. add 35 µL water onto the column 22. incubate for 1 min RT 23. centrifuge at 9000 rpm, 15 s RT 24. dry the samples in a SpeedVac --> dried cDNA In-Vitro-Transcription Preparations: thaw the 10× T7 reaction buffer thaw rNTP-Mix and mix well Compose the following mixture: 8 µL water 1/10 Vol. (2 µL) 10× T7 reaction buffer 150 nmol (8 µL) rNTP-Mix dissolve the dried cDNA in 18 µL of the mixture by vortexing and put on ice add 2 µL MegaScript T7 enzyme solution incubate for 3 h, 37 °C incubate for 10 min, 65 °C keep at 4 °C or on ice Purification with Qiagen RNeasy mini kit 1. add 6 µL 2-Mercaptoethanol per 1000 µL RLT 2. pipet 300 µL Ethanol absolute into a 1.5 mL tube and keep on ice 3. add 300 µL RLT + ME to the sample 4. transfer the mixture to the tube with ethanol and mix well by pipeting 5. apply the mixture to an RNeasy column 6. centrifuge 9000 rpm, 15 s, RT 7. discard the filtrate 8. add 600 µL RW1 onto the column 9. centrifuge 9000 rpm, 15 s, RT 10. discard the filtrate and receiver tube and place the column into a fresh receiver tube 11. add 500 µL RPE onto the column 12. centrifuge 9000 rpm, 15 s, RT 13. discard the filtrate 14. add another 500 µL RPE onto the column 15. centrifuge 9000 rpm, 15 s, RT 16. discard the filtrate 17. centrifuge at maximum speed, 1 min, RT 18. discard the receiver tube and place the column into a fresh RNase-free 1.5 mL tube 19. add 45 µL RNase-free water onto the column 20. incubate for 1 min, RT 21. centrifuge at 9000 rpm, 15 s, RT 22. incubate for 1 min, RT 23. centrifuge at 9000 rpm, 15 s, RT 24. take a 1:100 dilution of the sample for spectrophotometric measurement at 260 nm 25. determine the amount of RNA from the absorbance at 260 nm 26. samples can be stored a –20 °C --> cRNA Labeling Materials 1. RNase Out, 40 U/µL (Invitrogen, 10777-019) 2. Random-Nanomers N9, 1 µg/µL (Metabion) 3. SuperScript II RT RNase H – reverse transcriptase (Invitrogen, 18064-014) package contains: SuperScript II RT, 5× first strand buffer, 0,1M DTT 4. –C-dNTP-Mix: 10 mM dATP, 2 mM dCTP, 10 mM dGTP, 10 mM dTTP (Amersham Pharmacia Biotech, 272050, 272060, 272070, 272080) 5. Cy3-dCTP for Microarrays (Amersham Pharmacia Biotech, PA 53021) 6. Cy5-dCTP for Microarrays (Amersham Pharmacia Biotech, PA 55021) 7. 2,5 M NaOH 8. 2 M MOPS 9. 3 M Sodium acetate, pH 5,0 with glacial acetic acid 10. QiaQuick PCR Purification Kit (Qiagen, 28106) Protocol Preparations: pre-warm a heating block to 70 ° C thaw 5× first strand buffer, DTT und –C-dNTP-Mix and keep on ice don't work in bright light while usind Cy dyes! Make up the following reaction: 5 µg cRNA 20 U *) (0,5 µL *)) RNase Out adjust with RNase-free water to 8 µL; *) if the volume of cRNA exceeds 8,5 µL, add 40 U (1 µL) RNase Out and dry out in a SpeedVac 2 µg (2 µL) Random-Nanomers N9 incubate for 10 min, 70 ° C, chill on ice and spin down 1/5 Vol. (4 µL) 5× first strand buffer 2 nmol dCTP, 10 nmol dDTP (1 µL) –C-dNTP 200 nmol (2 µL) 0,1 M DTT 1,5 nmol (1,5 µL) Cy5-dCTP 200 U (1 µL) SuperScript II RT incubate for 2½ h 42 °C chill on ice or go to 4 °C add 2 µL 2,5 M NaOH incubate for EXACTLY 15 min, 37 ° C add 10 µL 2 M MOPS Purification with QIAquick PCR Purification Kit 1. add 5 µL 3 M Sodium acetate, pH5 to the sample 2. add 5 Vol. Puffer PB zugeben (usually 185 µL) 3. mix well 4. pipet the mixture onto a QiaQuick column 5. centrifuge at 9000 rpm, 15 s, RT 6. discard the filtrate 7. add 500 µL PE onto the column 8. centrifuge at 9000 rpm, 15 s, RT 9. discard the filtrate 10. add another 500 µL onto the column 11. centrifuge at 9000 rpm, 15 s, RT 12. discard the filtrate 13. centrifuge at maximum speed, 1 min 14. discard the filtrate and receiver tube 15. transfer the column into a fresh 1.5 mL tube 16. add 50 µL 1/5 EB onto the column 17. incubate for 1 min, RT 18. centrifuge at 9000 rpm, 15 s, RT 19. if any remains of dye are visible on the membrane, add another 50 µL onto the column 20. incubate for 1 min, RT 21. centrifuge at 9000 rpm, 15 s, RT 22. dry out in a SpeedVac (darken the SpeedVac with aluminum foil) 23. store the samples at –20 °C in the dark --> Cy5-labeled target
(Parameters: Amount of nucleic acid labeled = 5, Mass unit = Micro gram, Label used = Cy5, Amplification = none)
P-MTAB-33694
treatment protocol
1 micromolar of 6-Benzyl aminopurine was added to Arabidopsis seedlings at growth stage 1.00 in liquid culture and incubated for the time indicated under normal light. The seedlings were then transferred to liquid nitrogen.
P-MTAB-33693
growth protocol
20 mg of Arabidopsis seeds were surface-sterilized with chlorine gas, resuspended in 1 mL 0.1% Agar and distributed into 4 petri dishes containing 20 mL of the media. The plants were grown in a Percival AR-66L growth cabinet at 24 °C, 16 h day and 8 h night. Growth stage 1.00 was reached after 5 to 7 days.
(Parameters: time unit = minutes, min temperature = 24, temperature unit = C, media = 1/2MS, 1 g/L sucrose, 0.5 g/L MES, pH6)
P-MTAB-33701
nucleic acid extraction protocol
Materials: 1. 3 M Sodium acetate, pH 5 with glacial acetic acid 2. TRIzol-Reagent: 38% Phenol (in saturated buffer) 800 mM Guanidinium thiocyanate 400 mM Ammonium thiocyanate 100 mM Sodium acetate (from 3M stock, pH 5) 5% Glycerol prepare the solution daily in the required quantities; don't use old TRIzol! 3. High salt solution: 1,2 M Sodium chloride 800 mM Sodium citrate 4. Isopropanol 5. Chloroform-Isoamylalcohol 24:1 6. 75% Ethanol (non-denatured) 7. RNase-free water 8. liquid nitrogen 9. Arabidopsis tissue samples (100 – 200 mg in 2 mL tubes) Protocol: 1. grind 100 – 200 mg of Arabidopsis tissue samples under liquid nitrogen to a fine powder and put it into a 2 mL tube. Don't allow the sample to thaw! 2. add 1000 µL TRIzol 3. vortex until the sample is thawed completely and gives a homogenous mixture. From now on you can work at room tempereature. 4. incubate for 5 min, RT 5. centrifuge at 16.000 ×g, 5 min, 4 °C 6. transfer supernatant into a fresh 2 mL tube 7. add 400 µL Chloroform-Isoamylalcohol 24:1 8. vortex briefly or shake vigorously by hand until the mixture looks homogenous 9. incubate for 5 min, RT 10. centrifuge at 16.000 ×g, 15 min, 4 °C 11. transfer 700 µL of the upper phase to a fresh 1.5 mL tube, without taking anything of the two other phases (rather take less of the upper phase) 12. add 350 µL Isopropanol 13. add 350 µL high salt solution (mixture turns cloudy) 14. mix by repeated inversion until the mixture turns clear again 15. incubate for 10 min, RT 16. centrifuge at 12.000 ×g, 10 min, 4 °C 17. remove supernatant completely by pipeting (use yellow tip if necessary) 18. add 900 µL 75% Ethanol and vortex briefly 19. centrifuge at 7500 ×g, 5 min, 4 °C 20. remove supernatant completely by pipeting (use yellow tip if necessary) 21. add 900 µL 75% Ethanol and vortex briefly 22. centrifuge at 7500 ×g, 5 min, 4 °C 23. remove supernatant completely by pipeting (use yellow tip if necessary) 24. dry pellet at max. 60 ° C until it turns clear. Don't overdry the pellet! 25. dissolve pellet in 30 – 40 µL RNase-free water (incubation for 5 min, 60 ° C helps a lot) 26. take a 1:100 dilution in TE for photometric measurement at 260, 280, and 230 nm 27. calculate the amount of RNA and the ratios 260/280 and 260/230 28. take a sample of 1 µg total RNA for a test gel 29. perform an RNA cleanup with Qiagen RNeasy mini columns according to the manufacturer's instructions, including the optional on-column DNase digestion 30. the RNA is suitable for amplification by in-vitro transcription if R260/280 > 1,950, and R260/230 > 2,050, and no significant amount of ethidium bromide fluorescence remains in the gel slots, and the RNA is not degraded. Note that the ratios work only with TE buffered dilutions!
(Parameters: Extracted product = total_RNA, Amplification = none)