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==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 ===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 1ul 4ul Total 100ul 400ul 94C 3min -> 10 cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C Qiaquick column purification. tube 1 (shaked): 30ul * 257.1ng/ul tube 2 (CTRL): 30ul * 194.6ng/ul Second attempt (3-16-09): 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 (tube1 / tube2) 1ul/0.5ul 4ul/2ul Total 100ul 400ul 94C 3min -> 10 cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C Qiaquick column purification. tube 1 (shaked): 30ul * 87.2ng/ul tube 2 (CTRL): 30ul * 75.9ng/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) First attempt: Total 20ul tube 1(257.1ng/ul) 4ul tube 2 (194.6ng/ul) 6ul 10X NEBuffer 4 2ul 10X NEBuffer 4 2ul 1mM SAM(fresh) 2ul 1mM SAM(fresh) 2ul 2U/ul Mme I 8ul 2U/ul Mme I 8ul ddH2O 4ul ddH2O 2ul Second attempt: Total 40ul DNA 13ul 10X NEBuffer 4 4ul 1mM SAM(fresh) 6ul 2U/ul Mme I 8ul ddH2O 9ul 1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h MinElute column purify. Elute in 11ul EB. TBE gel result: [[Image:ZhangLab_2 2009-03-14 16hr 19min - TBE.jpg]] [[Image:ZhangLab_2 2009-03-17 10hr 59min-TBU.jpg]] ===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 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. TBE gel result: [[Image:ZhangLab_2 2009-03-18 10hr 45min.jpg]] ===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 65C for 25 minutes -> keep on ice. ===PCR of sequencig library=== Nick-translated DNA 10ul Solexa_PCR_up(100uM) 0.2ul Solexa_PCR_lo(100uM) 0.2ul 2xiProof master mix 50ul 50x SYBG 0.8ul ddH2O 35.2ul two tubes: product with cosmic probe and positive control 98C 30sec -> 10 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold. [[Image:3-18-09 PCR of solexa library.jpg]] red line: control blue line: PGP1-shaked Green line: PGP1-CTRL Nanodrop esult: PGP1-CTRL: 48.5ng/ul PGP1-shaked: 46ng/ul Purify with Qiaquick column. Elute in 30ul EB. TBE gel result: [[Image:ZhangLab_2 2009-03-18 15hr 20min.jpg]]
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