Alice:LabNotes/2009-3-13
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Construct Pair-end Solexa sequencing library
(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
2nd PCR amplification
AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40 system setup: x4 H2O 43.2ul 172.8ul 2x Master mix 50ul 200ul dUTP(1mM) 2ul 8ul AmpF6.3(10uM) 2ul 8ul AmpR6.3(10uM) 2ul 8ul 50x SYBG I 0.4ul 1.6ul template(19ng/ul) 0.1ul 0.4ul Total 100ul 400ul
94C 3min -> 9 cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C Qiaquick column purification. 30ul * 47ng/ul
Digestion with MmeI
(dUTP 250uM,167.3ng/ul of product will use the system below) Total 20ul DNA 6ul 10X NEBuffer 4 2ul 1mM SAM(fresh) 2ul 2U/ul Mme I 8ul ddH2O 2ul 1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h
however, since the concentration I obtained from previous is 47ng/ul, therefore the following is added: DNA 14ul SAM(1mM) 4ul NEBuffer 4 2ul 37C 2hr MinElute column purify. Elute in 11ul EB.
USER digestion (1/14/09)
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
positive control Total 25ul 25ul DNA 15ul 2ul (obtained from Ida: 20080801_spacer_107bp) dNTP 2.5ul 2.5ul 10xendrepair buffer 2.5ul 2.5ul enzyme 0.5ul 0.5ul ATP(10mM) 5ul 5ul H2O 13ul Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.
adapter ligation (1/15/09)
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 positive control negative control DNA 13ul 13ul ddH2O 13ul 100uM Solexa_1 adaptor 0.5ul 0.5ul 0.5ul 100uM Solexa_2_PE adaptor 0.5ul 0.5ul 0.5ul 2xQuickLiage buffer 15ul 15ul 15ul QuickLigase enzyme(NEB) 1ul 1ul 1ul extra ATP(10mM) 2.5ul 2.5ul 2.5ul Keep at room temperature (~25C) for 10 minutes. Purify with Minelute column. Elute in 20ul H2O.
PAGE selection 150-175bp. Ethanol precipitation. Elute in 15ul ddH2O. 2D gel result:
Nick-translation (1/19/09)
set up the ligation system: Total 20ul DNA 15ul 10x ThermoPol buffer 2ul 10mM dNTP 0.4ul 1mg/ml BSA 2ul Bst polymerase(8U/ul) 1ul two tubes: product with cosmic probe and positive control
65C for 25 minutes -> keep on ice.
PCR of sequencig library
Nick-translated DNA 10ul Solexa_PCR_upH(10uM) 2ul Solexa_PCR_PE_loH(10uM) 2ul 2xiProof master mix 50ul 50x SYBG 0.8ul ddH2O 35.2ul two tubes: product with cosmic probe and positive control 98C 30sec -> 17 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
Purify with Qiaquick column. Elute in 30ul EB. TBE gel result: