Alice:LabNotes/2009-9-15: Difference between revisions
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Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 20ul H2O. | Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 20ul H2O. | ||
PAGE size selection | PAGE size selection | ||
[[Image:ZhangLab_2 2009-09-16 12hr 47min.jpg]] | |||
[[Image:ZhangLab_2 2009-09-16 12hr 48min.jpg]] | |||
===adapter ligation=== | ===adapter ligation=== |
Revision as of 20:43, 18 September 2009
Single-end solexa sequencing library
Materials
use previously captured samples capture reaction setup can be found under LabNotes 8-15-09 and 8-16-09 samples to be used: D3(6&7 subsets of NA12878), DF6-9-9, foreskin, ips(PGP1-ips), Cvi(CV-ips), CVf(CV-fibroblast)
Procedure
Reagent setup: (Pair-end sequencing library construction) Solexa_1 adaptor and Solexa_2_PE adaptor will be used for PCR at the last step use these primers: Solexa_PCR_PE_loH & Solexa_PCR_upH (single-end sequencing library construction) Solexa_1 adaptor and Solexa_2 adaptor will be used for PCR at the last step use these primers: Solexa_PCR_up Solexa_PCR_lo
PCR re-amplification
AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40 system setup: x4 H2O 46ul 184ul 2x KAPA Master mix 50ul 200ul dUTP(1mM) 4ul 16ul AmpF6.3NH2(100uM) 0.4ul 8ul AmpR6.3NH2(100uM) 0.4ul 8ul template 0.5ul 2ul 95C 30sec -> (95C 3sec -> 60C 30sec) x 3 -> 60C 2min -> 15C hold.
Qiaquick column purification and elute in 30ul EB. Nanodrop: D3: 19.2ng/ul DF: 33.3ng/ul fs: 17.7ng/ul ips: 13ng/ul cvi: 23.7ng/ul cvf: 12.2ng/ul
Digestion with MmeI
(dUTP 250uM,167.3ng/ul of product will use 6ul of DNA along with 2ul of 1mM SAM and 8ul of 2U/ul MmeI) DF & cvi ips & cvf D3 & fs DNA 30ul 30ul 30ul 10X NEBuffer 4 5ul 4ul 5ul 1mM SAM(fresh) 6ul 3ul 4ul 2U/ul Mme I 8ul 3ul 5ul ddH2O 1ul - 6ul 1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h
MinElute column purify. Elute in 11ul EB.
USER digestion
DNA 10ul USER 3ul total 13ul 37C 4hr
S1 nuclease digestion
10 x S1 nuclease buffer: 2ul DNA after USER digestion: 13ul S1 nuclease (10U/ul): 1ul ddH2O 4ul
37C 10mins. Minelute cloumn purify. Elute in 16ul H2O.
end repair
Total 25ul DNA 15ul dNTP 5ul 10xendrepair buffer 2.5ul enzyme 1ul ATP(10mM) 5ul Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 20ul H2O. PAGE size selection File:ZhangLab 2 2009-09-16 12hr 47min.jpg File:ZhangLab 2 2009-09-16 12hr 48min.jpg
adapter ligation
adaptor construction: add 10ul Solexa_2_PE_up and 10ul Solexa_2_PE_lo_noP. 10ul Solexa_2_upNH2 and 10ul Solexa_2_lo_noP. 10ul Solexa_1_upNH2 and 10ul Solexa_1_lo_noP. 95C 5mins -> 65C 30mins -> 4C.
total 30ul DNA 13ul 100uM Solexa_1 adaptor 1ul 100uM Solexa_2 adaptor 1ul 2xQuickLiage buffer 15ul QuickLigase enzyme(NEB) 1ul extra ATP(10mM) 2.5ul Keep at room temperature (~25C) for 10 minutes. Purify with Minelute column. Elute in 10ul H2O.
Nick-translation
DNA 10ul 10x ThermoPol buffer 1.2ul 10mM dNTP 0.5ul Bst polymerase(8U/ul) 0.5ul
65C for 12 minutes
PCR of sequencig library
Nick-translated DNA 6ul Solexa_PCR_up(100uM) 0.2ul Solexa_PCR_lo(100uM) 0.2ul 2xiProof master mix 50ul 50x SYBG 0.2ul ddH2O 44ul 98C 30sec -> 7-8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold. PAGE size selection 175-225bp