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