Jie:LabNotes/CpgSeq/2009-8-7

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Revision as of 18:17, 16 August 2009 by >Sam Chiang
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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. Quantify the e-gel selected amplicon and mix the cpg97k_A and cpg97k_B amplicons  with 1:1 ratio. 

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.
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                                          0.5ul/each for purified cpg97k
Total                                               100ul      1400ul