Jie:LabNotes/CpgSeq/2009-5-20
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bisulfite conversion of PGP samples
sample | sample concentration | sample volumn | ddH2O | conversion reagents | conversed DNA concentration and volumn | |
09_1 | NA21687*1 062507 | 241.1ng/ul x 1 tubes | 8ul | 12ul | 130ul | 95.4ng/ul x 10ul |
09_2 | NA21781*1 102307 | 240ng/ul x 1 tubes | 8ul | 12ul | 130ul | 120.2ng/ul x 10ul |
09_3 | NA21833*1 101807 | 253.9ng/ul x 1 tube | 8ul | 12ul | 130ul | 121.5ng/ul x 10ul |
09_4 | NA21660*1 053007 | 243.2ng/ul x 1 tube | 8ul | 12ul | 130ul | 120.3ng/ul x 10ul |
09_5 | NA21731*1 101807 | 217.4ng/ul x 1 tube | 10ul | 10ul | 130ul | 127.3ng/ul x 10ul |
09_6 | NA21846*1 100407 | 186.5ng/ul x 1 tube | 11ul | 9ul | 130ul | 151.3ng/ul x 10ul |
09_7 | NA21070*1 100406 | 329.7ng/ul x 1 tube | 6ul | 14ul | 130ul | 311.2ng/ul x 10ul |
Jurkat gDNA | 140ng/ul x 3 tube | 14ul | 6ul | 130ul | 255.6ng/ul x 20ul |
- 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