Jie:LabNotes/CpgSeq/2009-8-7: Difference between revisions
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>Sam Chiang No edit summary |
>Sam Chiang No edit summary |
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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 | 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 | ||
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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. | 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.