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

From ZhangLabWiki
Jump to navigation Jump to search
>Jie deng
No edit summary
>Sam Chiang
No edit summary
Line 38: Line 38:
|-
|-
}
}
===PCR amplification with AmpF6.3NH2/AmpR6.3NH2 and dUTP:dNTP 1:40===
I did the dUTP_PCR with template from the mixture captured targets, the Qiaquick purified captured targets of 97k, and the directed 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                      0.25ul/each for cpg30k mixture, 0.05ul/each for purified cpg97k, 1ul/each for direct cpg97k. 
Total                                              100ul      1400ul

Revision as of 17:40, 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

}

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

I did the dUTP_PCR with template from the mixture captured targets, the Qiaquick purified captured targets of 97k, and the directed 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 0.25ul/each for cpg30k mixture, 0.05ul/each for purified cpg97k, 1ul/each for direct cpg97k. Total 100ul 1400ul

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