Jie:LabNotes/CpgSeq/2009-1-22

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6th 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
21 PGP1_iPS1 50ng/ul x 3 tubes 20ul 0ul 130ul 179.1ng/ul x 10ul 1.97/1.72
22 PGP1_L 293ng/ul x 3 tubes 6.8ul 13.2ul 130ul 368.7ng/ul x 20ul 2.13/2.04
23 PGP3_L 242ng/ul x 3 tubes 6.8ul 13.2ul 130ul 238.6ng/ul x 20ul 2.07/1.89
24 PGP9_L 249ng/ul x 3 tubes 6.8ul 13.2ul 130ul 223.7ng/ul x 20ul 2.05/1.89
25 PGP1F.2 25ng/ul x 4 tubes 20ul 0ul 130ul 84ng/ul x 10ul 1.67/1.2
26 PGP9F.2 14ng/ul x 4 tube 20ul 0ul 130ul 35.1ng/ul x 10ul 1.2/0.66


  • 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
21 PGP1_iPS1 179.1ng/ul x 10ul 1ul 1+1+1+6ul 1+1.5+6.5 3+1.5+4.5 2+1.5+5.5
22 PGP1_L 368.7ng/ul x 10ul 1ul 1+1+1+6ul 1+1.5+6.5 3+1.5+4.5 2+1.5+5.5
23 PGP3_L 238.6ng/ul x 10ul 1ul 1+1+1+6ul 1+1.5+6.5 3+1.5+4.5 2+1.5+5.5
24 PGP9_L 223.7ng/ul x 10ul 1ul 1+1+1+6ul 1+1.5+6.5 3+1.5+4.5 2+1.5+5.5
25 PGP1F.2 84ng/ul x 10ul 1ul 7+1+1+0ul
26 PGP9F.2 35.1ng/ul x 10ul 1ul 7+1+1+0ul
positive control 200ng/ul 1ul 1+1+1+6ul 1+1.5+6.5 3+1.5+4.5 2+1.5+5.5
negative control 0 1ul 1+1+1+6ul 1+1.5+6.5 3+1.5+4.5 2+1.5+5.5


 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.

PCR(08/16/2008)

                                      x8
   Template                10ul      
   2X iProof Mastermix     50ul      400ul
   AmpF6.2SoL (10uM)        4ul       32ul
   AmpR6.2SoL (10uM)        4ul       32ul   
   50X SYBG I             0.8ul      6.4ul
   H2O                   31.2ul    249.6ul
   

98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold.