Alice:LabNotes/2009-1-13
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Construct Solexa sequencing library
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
step2. 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 22ul EB.
step3. USER digestion
DNA 10ul USER 3ul total 13ul 37C 4hr
step4. 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.
step5. end repair
positive control Total 25ul 25ul DNA 15ul 2ul (obtained from Ida: 20080801_spacer_107bp) dNTP 2.5ul 2.5ul dATP 2.5ul 2.5ul 10xendrepair buffer 2.5ul 2.5ul enzyme 0.5ul 0.5ul extra ATP(10mM) 2.5ul 2.5ul H2O 13ul Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.
step6. adapter ligation
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 cilumn. Elute in 20ul H2O.
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) 2ul 6ul Solexa_PCR_lo(10uM) 2ul 6ul 2xiProof master mix 50ul 150ul 50x SYBG 0.8ul 2.4ul ddH2O 35.2ul 106.6ul 98C 30sec -> 12 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
Purify with Qiaquick column. Elute in 30ul EB.