Jie:LabNotes/CpgSeq/2009-8-7
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 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