Jie:LabNotes/CpgSeq/2009-5-29: Difference between revisions

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   50X SYBG I            0.8ul        8ul
   50X SYBG I            0.8ul        8ul
   H2O                  36.2ul      362ul
   H2O                  36.2ul      362ul


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

Revision as of 17:02, 2 June 2009

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