Jie:LabNotes/CpgSeq/2009-5-29

From ZhangLabWiki
Revision as of 17:02, 2 June 2009 by KunZhang (talk | contribs)
Jump to navigation Jump to search

Bisulfite conversion of PGP DNA for cpg30k ASM validation

No sample sample concentration sample volumn ddH2O conversion reagents conversed DNA concentration and volumn
20 GM20431 256ng/ul x 1 tube 8ul 12ul 130ul 56.1ng/ul x 10ul
21 PGP1_iPS1 50ng/ul x 1 tubes 10ul 10ul 130ul 40.7ng/ul x 10ul
22 PGP1_L 293ng/ul x 1 tubes 6.8ul 13.2ul 130ul 191.6ng/ul x 10ul
23 PGP3_L 242ng/ul x 1 tubes 6.8ul 13.2ul 130ul 114.7ng/ul x 10ul
24 PGP9_L 249ng/ul x 1 tubes 6.8ul 13.2ul 130ul 106.5ng/ul x 10ul
09_8 DF6-9-9_P16(4) 504.8ng/ul x 3 tubes 4ul 16ul 130ul 285.7ng/ul x 10ul
09_9 foreskin 583ng/ul x 2 tube 4ul 16ul 130ul 212.5ng/ul x 10ul


  • 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 reaction

No sample sample concentration 10xLigase buffer template+cpg30k(10ng/ul,2009-05-29 by Alan)+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 template+Jan09#2(17ng/ul_05/20) vol+H2O
09_8 DF6-9-9_P16(4) 285.7ng/ul x 10ul 1ul 1+3+1+3ul 1+1.5+6.5 3+1.5+4.5 2+1.5+5.5 2+5ul+2ul
09_9 foreskin 212.5ng/ul x 10ul 1ul 1+3+1+3ul 1+1.5+6.5 3+1.5+4.5 2+1.5+5.5 2+5ul+2ul
positive control Jurkat bis-gDNA 200ng/ul 1ul 1+3+1+6ul
negative control 0 1ul 0+3+1+6ul

dNTP concentration 10mM(NEB)

                                                             ddH2O  Ligase Buffer   Ligase  Amplitaq dNTP           
2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP:  0.5ul    0.1ul    0.1ul       0.2ul    (10mM)0.1ul 
                                                          x60  30ul     6ul       6ul       12ul      6ul

PCR with AmpF/R6.2Sol and iProof

                                     x10
  Template                 5ul      
  2X iProof Mastermix     50ul      500ul
  AmpF6.2SoL (10uM)        4ul       40ul
  AmpR6.2SoL (10uM)        4ul       40ul   
  50X SYBG I             0.8ul        8ul
  H2O                   36.2ul      362ul


==PCR amplification with AmpF6.3NH2/AmpR6.3NH2 and dUTP:dNTP 1:40

reaction system                                                 x14     x2
H2O                                                42.6ul     596.4ul    
2x Master mix                                        50ul      700ul      
dUTP(1mM)                                             2ul       28ul       
AmpF6.3NH2(10uM)                                      2ul       28ul       
AmpR6.3NH2(10uM)                                      2ul       28ul     
50x SYBG I                                          0.4ul      5.6ul    
template(4ng/ul)                                    0.5ul       2 uleach     
Total                                               100ul      1400ul     
94C 3min -> 9cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C