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Jie:LabNotes/CpgSeq/2009-9-28
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==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 x2 Total 80ul PCR 48ul 10X NEBuffer 4 8ul 16 1mM SAM(fresh) 16ul 32 2U/ul Mme I 8ul 16 1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h. Qiaquick column purification. Elute in 50ul EB. 28ul samples are used for endrepair and 22ul are used for ligation directly. [[Image:20090928_after MmeI.jpg]]20090928_after MmeI ===endrepair with enzymatic end-repair kit=== x2 28 ul DNA 4 ul 10X End-Repair Buffer 8 4 ul dNTP Mix 8 4 ul End-Repair Enzyme Mix 8 40 ul Total reaction volume RT for 30 min. Qiaquick column purification. Elute in 30ul EB. ===A tail addition=== x2 Blunt-ended DNA 30ul 30 each 10X Klenow buffer 4ul 8 1mM dATP 6ul 12 Klenow fragment (exo-)10U/ul 2ul 4 37C 30min, purified with MinElute columns, eluted with 12ul EB. ===adaptor ligation=== x8 DNA 10ul 2x QuickLigase buffer (enzymatic) 15ul 120 20uM Adaptor oligo mix 3ul 24 T4 DNA QuickLigase (enzymatic) 2ul 16 Incubate at RT for 15 minutes. Purified with Minelute columns, eluted with 10ul EB. TBU gel size selection: 200bp, 225-275bp, >275bp. ===PCR=== [[Image:20090930_lib.jpg]]20090930_lib ===quantification with Q-PCR=== CViPS: 27.37nM; CVfibroblast: 19.6nM.
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