Jie:LabNotes/CpgSeq/2009-9-28: Difference between revisions
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==Exom-samples Sequencing library construction without shearing== | ==Exom-samples Sequencing library construction without shearing== | ||
I constructed the library with captured product found under Alice's labnotes 8/15/09 and 8/16/09 | |||
===Pool the products together=== | |||
pooled all the subsets according ALice's labnote on 8/20/09 | |||
CV-ips 1 CV-ips 2 CV-ips 3 CV-ips 4 | |||
concnetration(ng/ul): 31.3ng/ul 10.2ng/ul 25ng/ul 12ng/ul | |||
volume needed (ul): 2ul 3ul 1.8ul 3.8ul | |||
-------------------------------------------------------------------------- | |||
total (ng): 62.6ng 31.3ng 46ng 45ng | |||
CV-fb 1 CV-fb 2 CV-fb 3 CV-fb 4 | |||
concnetration(ng/ul): 12.3ng/ul 6.1ng/ul 11.7ng/ul 11.2ng/ul | |||
volume needed (ul): 3ul 3ul 2.3ul 2.4ul | |||
-------------------------------------------------------------------------- | |||
total (ng): 36.9ng 18.5ng 27ng 26.5ng | |||
===PCR with Amp6.3NH2 primer=== | ===PCR with Amp6.3NH2 primer=== | ||
I did the PCR reamplification with Amp6.3NH2 primers. 400ul for each sample. | I did the PCR reamplification with Amp6.3NH2 primers. 400ul for each sample. | ||
reaction system x4 | reaction system x4 x2 | ||
H2O 45ul 180ul | H2O 45ul 180ul 360 | ||
2x iProof Mastermix 50ul 200ul | 2x iProof Mastermix 50ul 200ul 400 | ||
AmpF6.3NH2(10uM) 2ul 8ul | AmpF6.3NH2(10uM) 2ul 8ul 16 | ||
AmpR6.3NH2(10uM) 2ul 8ul | AmpR6.3NH2(10uM) 2ul 8ul 16 | ||
50x SYBG I 0.4ul 1.6ul | 50x SYBG I 0.4ul 1.6ul 3.2 | ||
template 1ul/each for purified amplicons | template 1ul/each for purified amplicons | ||
Total 100ul 800ul | Total 100ul 800ul |
Revision as of 23:24, 28 September 2009
Exom-samples Sequencing library construction without shearing
I constructed the library with captured product found under Alice's labnotes 8/15/09 and 8/16/09
Pool the products together
pooled all the subsets according ALice's labnote on 8/20/09
CV-ips 1 CV-ips 2 CV-ips 3 CV-ips 4 concnetration(ng/ul): 31.3ng/ul 10.2ng/ul 25ng/ul 12ng/ul volume needed (ul): 2ul 3ul 1.8ul 3.8ul -------------------------------------------------------------------------- total (ng): 62.6ng 31.3ng 46ng 45ng
CV-fb 1 CV-fb 2 CV-fb 3 CV-fb 4 concnetration(ng/ul): 12.3ng/ul 6.1ng/ul 11.7ng/ul 11.2ng/ul volume needed (ul): 3ul 3ul 2.3ul 2.4ul -------------------------------------------------------------------------- total (ng): 36.9ng 18.5ng 27ng 26.5ng
PCR with Amp6.3NH2 primer
I did the PCR reamplification with Amp6.3NH2 primers. 400ul for each sample. reaction system x4 x2 H2O 45ul 180ul 360 2x iProof Mastermix 50ul 200ul 400 AmpF6.3NH2(10uM) 2ul 8ul 16 AmpR6.3NH2(10uM) 2ul 8ul 16 50x SYBG I 0.4ul 1.6ul 3.2 template 1ul/each for purified amplicons Total 100ul 800ul 98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 ->72C 3min. Purify with Qiaquick column. Elute in 100ul EB.
MmeI digestion
Digestion with MmeI x7 Total 50ul PCR 28ul 10X NEBuffer 4 5ul 35 1mM SAM(fresh) 10ul 70 2U/ul Mme I 8ul 56
1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h. Qiaquick column purification. Elute in 30ul EB.