Jie:LabNotes/CpgSeq/2009-5-29: Difference between revisions
Jump to navigation
Jump to search
No edit summary |
No edit summary |
||
Line 10: | Line 10: | ||
| align="center" style="background:#f0f0f0;"|'''conversion reagents''' | | align="center" style="background:#f0f0f0;"|'''conversion reagents''' | ||
| align="center" style="background:#f0f0f0;"|'''conversed DNA concentration and volumn''' | | align="center" style="background:#f0f0f0;"|'''conversed DNA concentration and volumn''' | ||
|- | |- | ||
|20||GM20431||256ng/ul x 1 tube||8ul||12ul||130ul|| | |20||GM20431||256ng/ul x 1 tube||8ul||12ul||130ul||56.1ng/ul x 10ul | ||
|- | |- | ||
|21||PGP1_iPS1||50ng/ul x 1 tubes||10ul||10ul||130ul|| | |21||PGP1_iPS1||50ng/ul x 1 tubes||10ul||10ul||130ul||40.7ng/ul x 10ul | ||
|- | |- | ||
|22||PGP1_L||293ng/ul x 1 tubes||6.8ul||13.2ul||130ul|| | |22||PGP1_L||293ng/ul x 1 tubes||6.8ul||13.2ul||130ul||191.6ng/ul x 10ul | ||
|- | |- | ||
|23||PGP3_L||242ng/ul x 1 tubes||6.8ul||13.2ul||130ul|| | |23||PGP3_L||242ng/ul x 1 tubes||6.8ul||13.2ul||130ul||114.7ng/ul x 10ul | ||
|- | |- | ||
|24||PGP9_L||249ng/ul x 1 tubes||6.8ul||13.2ul||130ul|| | |24||PGP9_L||249ng/ul x 1 tubes||6.8ul||13.2ul||130ul||106.5ng/ul x 10ul | ||
|- | |- | ||
|09_8||DF6-9-9_P16(4)||504.8ng/ul x 3 tubes||4ul||16ul||130ul|| | |09_8||DF6-9-9_P16(4)||504.8ng/ul x 3 tubes||4ul||16ul||130ul||285.7ng/ul x 10ul||1.67/1.2 | ||
|- | |- | ||
|09_9||foreskin||583ng/ul x 2 tube||4ul||16ul||130ul|| | |09_9||foreskin||583ng/ul x 2 tube||4ul||16ul||130ul||212.5ng/ul x 10ul | ||
|- | |- | ||
|} | |} |
Revision as of 21:44, 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 | |
20 | GM20431 | 256ng/ul x 1 tube | 8ul | 12ul | 130ul | 56.1ng/ul x 10ul | |
21 | PGP1_iPS1 | 50ng/ul x 1 tubes | 10ul | 10ul | 130ul | 40.7ng/ul x 10ul | |
22 | PGP1_L | 293ng/ul x 1 tubes | 6.8ul | 13.2ul | 130ul | 191.6ng/ul x 10ul | |
23 | PGP3_L | 242ng/ul x 1 tubes | 6.8ul | 13.2ul | 130ul | 114.7ng/ul x 10ul | |
24 | PGP9_L | 249ng/ul x 1 tubes | 6.8ul | 13.2ul | 130ul | 106.5ng/ul x 10ul | |
09_8 | DF6-9-9_P16(4) | 504.8ng/ul x 3 tubes | 4ul | 16ul | 130ul | 285.7ng/ul x 10ul | 1.67/1.2 |
09_9 | foreskin | 583ng/ul x 2 tube | 4ul | 16ul | 130ul | 212.5ng/ul x 10ul |
- 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