Jie:LabNotes/CpgSeq/2008-7-31
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sequencing library construction
2nd PCR amplification with AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40
reaction system x4 x3 H2O 43.2ul 172.8ul 518.4 2x Master mix 50ul 200ul 600 dUTP(1mM) 2ul 8ul 24 AmpF6.3(10uM) 2ul 8ul 24 AmpR6.3(10uM) 2ul 8ul 24 50x SYBG I 0.4ul 1.6ul 4.8 template(18.4ng/ul) 0.1ul 0.4ul 1.2 Total 100ul 400ul 2400
94C 3min -> 13cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C Bioneer column purification. Yield:167ng/ul x60ul
step2. Digestion with MmeI
dUTP 250uM,167.3ng/ul x2 Total 20ul DNA 6ul 12 10X NEBuffer 4 2ul 4 1mM SAM(fresh) 2ul 4 2U/ul Mme I 8ul 16 ddH2O 2ul 4 1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h MinElute column purify. Elute in 11ul EB.
step3. USER digestion
DNA 10ul USER 3ul total 13ul
37C 12h.
step4. S1 nuclease digestion
x2 10 x S1 nuclease buffer: 2ul 4ul DNA after USER digestion: 13ul 26ul S1 nuclease (10U/ul): 1ul 2ul ddH2O 4ul 8ul
37C 10mins. Minelute cloumn purify. Elute in 18ul H2O.
step5. end repair
Total 25ul DNA 17ul dNTP 2.5ul dATP 2.5ul 10xendrepair buffer 2.5ul enzyme 0.5ul Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.
step6. adapter ligation
total 30ul control DNA 13ul ddH2O 13ul 100uM Solexa_1 adaptor 0.5ul 0.5ul 100uM Solexa_2 adaptor 0.5ul 0.5ul 2xQuickLiage buffer 15ul 15ul QuickLigase enzyme(NEB) 1ul 1ul
Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul EB.
PAGE selection 150-175bp. Ethanol precipitation. Elute in 15ul ddH2O.
step7. Nick-translation
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.
step8. PCR of sequencig library
x3 Nick-translated DNA 10ul Solexa_PCR_up(10uM) 1ul 3ul Solexa_PCR_lo(10uM) 1ul 3ul 2xiProof master mix 25ul 75ul 50x SYBG 0.4ul 1.2ul ddH2O 12.6ul 37.8ul 98C 30sec -> 13 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
Purify with Qiaquick column. Elute in 30ul EB. dUTP250uM: 12.8ng/ul dUTP100uM: 22.5ng/ul