Jie:LabNotes/CpgSeq/2008-8-29
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3rd Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold kit
- 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:
sample | sample concentration | sample volumn | ddH2O | conversion reagents | conversed DNA concentration and volumn | 260:280/260:230 | |
1 | BJ | 178ng/ul | 11.2ul | 8.8ul | 130ul | ng/ul x 10ul | 2.18/2.97 |
2 | BJ_iPS_11 | 140ng/ul | 14.3ul | 5.7ul | 130ul | ng/ul x 10ul | 2.19/2.91 |
3 | BJ_iPS_12 | 40ng/ul | 20ul | 0ul | 130ul | ng/ul x 10ul | |
9 | Hybrid2 | 200ng/ul | 10ul | 10ul | 130ul | ng/ul x 10ul | 2.3/3.0 |
Jurkat | 200ng/ul x 2 tube | 10ul | 10ul | 130ul | ng/ul x 20ul |