Jie:LabNotes/CpgSeq/2009-5-29: Difference between revisions
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| align="center" style="background:#f0f0f0;"|'''260:280/260:230''' | | align="center" style="background:#f0f0f0;"|'''260:280/260:230''' | ||
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| ||GM20431||256ng/ul x 1 tube||8ul||12ul||130ul|| | |20||GM20431||256ng/ul x 1 tube||8ul||12ul||130ul|| | ||
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|21||PGP1_iPS1||50ng/ul x 1 tubes||10ul||10ul||130ul||179.1ng/ul x 10ul||1.97/1.72 | |21||PGP1_iPS1||50ng/ul x 1 tubes||10ul||10ul||130ul||179.1ng/ul x 10ul||1.97/1.72 |
Revision as of 16:56, 29 May 2009
Bisulfite conversion of PGP DNA for cpg30k ASM validation
No | sample | sample concentration | sample volumn | ddH2O | conversion reagents | conversed DNA concentration and volumn | 260:280/260:230 |
20 | GM20431 | 256ng/ul x 1 tube | 8ul | 12ul | 130ul | ||
21 | PGP1_iPS1 | 50ng/ul x 1 tubes | 10ul | 10ul | 130ul | 179.1ng/ul x 10ul | 1.97/1.72 |
22 | PGP1_L | 293ng/ul x 1 tubes | 6.8ul | 13.2ul | 130ul | 368.7ng/ul x 20ul | 2.13/2.04 |
23 | PGP3_L | 242ng/ul x 1 tubes | 6.8ul | 13.2ul | 130ul | 238.6ng/ul x 20ul | 2.07/1.89 |
24 | PGP9_L | 249ng/ul x 1 tubes | 6.8ul | 13.2ul | 130ul | 223.7ng/ul x 20ul | 2.05/1.89 |
09_8 | DF6-9-9_P16(4) | 504.8ng/ul x 3 tubes | 4ul | 16ul | 130ul | 84ng/ul x 10ul | 1.67/1.2 |
09_9 | foreskin | 583ng/ul x 2 tube | 4ul | 16ul | 130ul | 35.1ng/ul x 10ul | 1.2/0.66 |
- Add 900ul ddH2O, 300ul M-Dilution Buffer and 50ul M-Dissolving Buffer to a tube of CT Conversion Reagent.
- Add 130ul of CT conversion reagent to samples.
- 98C 10min -> 64C 150min -> 4c overnight.
- Add 600ul M-Binding buffer to spin columns.
- Add the converted samples to the columns, close the gap and mix by inverting several times.
- Spin at 15,000rpm for 30sec.
- Add 100ul M-Wash Buffer, spin for 30 sec, discard flow-through.
- Add 200ul M-Desulphonation Buffer, wait for 20min, spin at 15,000 rpm for 30sec.
- Add 200ul M-Wash Buffer, spin for 30 sec.
- Place the columns into 1.5ml tubes, add 10ul M-Elution Buffer. Wait for 1min, spin at 15,000rpm for 30sec.
- Measure the DNA with Nanodrop:
RNA