Jie:LabNotes/CpgSeq/2008-8-29

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Revision as of 16:20, 29 August 2008 by >Jie deng
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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:
RNA
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