Jie:LabNotes/CpgSeq/2008-10-10

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Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold kit

sample sample concentration sample volumn ddH2O conversion reagents conversed DNA concentration and volumn 260:280/260:230
2 BJ_iPS_11 140ng/ul x 2 tubes 14.3ul 5.7ul 130ul 65.6ng/ul x 20ul 2.19/2.91
16 IMB90_2 50ng/ul 20ul 0ul 130ul 22.7ng/ul x 10ul 2.05/2.78
17


  • 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

set up the capture system

sample sample concentration 10xLigase buffer template+cpg30k(50.7ng/ul)+suppressor(8nM each)vol+H2O template+v2.1(21ng/ul) vol+H2O template+v2.2(21ng/ul) vol+H2O template+v2.3(20ng/ul) vol+H2O
2 BJ_iPS_11 65.6ng/ul x 20ul 1ul 2+1+1+5ul 3+1.5+4.5 5+1.5+2.5 4+1.5+3.5
16 IMB90 22.9ng/ul x 30ul 1ul 4+1+1+3ul
17 positive control 200ng/ul 1ul 1+1+1+6ul 1+1.5+6.5 2+1.5+5.5 1.5+1.5+6
negative control 0 1ul 1+1+1+6ul 1+1.5+6.5 2+1.5+5.5 1.5+1.5+6


 dNTP concentration 10mM(NEB)
                                                                      ddH2O     Ligase Buffer   Ligase     Amplitaq    dNTP           
 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP:      0.58ul         0.1ul       0.1ul       0.2ul    (10mM)0.02ul 
                                                           x300       174ul           30ul        30ul        60ul      6ul
 95c 10min -> 55C 16h ->add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP) -> 55C 4h -> 
9 cycels (95C 1min -> 55C for 4h) -> 95C 1min -> 37C 1min ->
add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold.