Jie:LabNotes/CpgSeq/2009-8-7: Difference between revisions

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>Sam Chiang
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>Sam Chiang
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  98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 ->(98C 10S -> 72C 20S) x 8 ->72C 3 min -> 15C hold.  
  98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 ->(98C 10S -> 72C 20S) x 8 ->72C 3 min -> 15C hold.  
  Qiaquick purification and e-gel size selection. Quantify the e-gel selected amplicon and mix the cpg97k_A and cpg97k_B amplicons  with 1:1 ratio.  
  Qiaquick purification and e-gel size selection.  


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


  I did the dUTP_PCR with template from the Qiaquick purified captured targets of 97k.
  I did the dUTP_PCR with template from the Qiaquick purified captured targets of 97k. For each targets, I did 200ul PCR reaction.
  reaction system                                                x8     
  reaction system                                                x8     
  H2O                                                42.6ul    340.8ul     
  H2O                                                42.6ul    340.8ul     
Line 61: Line 61:
  AmpR6.3NH2(10uM)                                      2ul      16ul     
  AmpR6.3NH2(10uM)                                      2ul      16ul     
  50x SYBG I                                          0.4ul      3.2ul     
  50x SYBG I                                          0.4ul      3.2ul     
  template                                          0.5ul/each for purified cpg97k
  template                                          0.25ul/each for e-gel purified cpg97k
  Total                                              100ul      1400ul
  Total                                              100ul      1400ul
Purify with qiaquick column. Quantify with nanodrop and mix them with 1:1 ratio.

Revision as of 19:36, 16 August 2009

breast cancer patient peripheral blood DNA sample capture by cpg97k

gDNA extraction from blood

I used the Qiagen FlexiGene DNA kit to exact the DNA from blood. yield:
05192A18: 83.5ng/ul x 100ul;
05192B09: 72.7ng/ul x 100ul

Bisulfite conversion of patient DNA

No sample sample concentration sample volumn ddH2O conversion reagents conversed DNA concentration and volumn
05192A18 83.5ng/ul x 1 tube 20ul 0ul 130ul 148.2ng/ul x 10ul
05192B09 72.7ng/ulx 1 tubes 20ul 0ul 130ul 127.8ng/ul x 10ul


cpature by cpg97k

No sample sample concentration 10xLigase buffer template+cpg97k_A(60ng/ul_08/10) vol+suppress oligo+H2O template+cpg97k_B(60ng/ul_08/10) vol+suppressor oligo+H2O
05192A18 148.2ng/ul 1ul 3+1.5ul+1ul+3.5ul 3+1.5ul+1ul+3.5ul
05192B09 127.8ng/ul 1ul 3+1.5ul+1ul+3.5ul 3+1.5ul+1ul+3.5ul
PCR
Template                15ul        x4
2X iProof Mastermix     50ul     
AmpF6.3SoL (10uM)        4ul       
AmpR6.3SoL (10uM)        4ul          
50X SYBG I             0.4ul      
H2O                   26.6ul    
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 ->(98C 10S -> 72C 20S) x 8 ->72C 3 min -> 15C hold. 
Qiaquick purification and e-gel size selection. 

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

I did the dUTP_PCR with template from the Qiaquick purified captured targets of 97k. For each targets, I did 200ul PCR reaction.
reaction system                                                 x8     
H2O                                                42.6ul     340.8ul    
2x Master mix                                        50ul      400ul      
dUTP(1mM)                                             2ul       16ul       
AmpF6.3NH2(10uM)                                      2ul       16ul       
AmpR6.3NH2(10uM)                                      2ul       16ul     
50x SYBG I                                          0.4ul      3.2ul    
template                                          0.25ul/each for e-gel purified cpg97k
Total                                               100ul      1400ul
Purify with qiaquick column. Quantify with nanodrop and mix them with 1:1 ratio.