Jie:LabNotes/CpgSeq/2008-9-12
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parallel sequencing library construction for No_1(BJ) and No_9(Hybrid_2)[edit]
Use the captured PCR mix of No_1 (BJ) and No_9 (Hybrid_2) as the templates. refer to LabNotes on [1]
2nd PCR amplification with AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40[edit]
reaction system x4 H2O 42.6ul 170.4ul 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(4ng/ul) 0.1ul 0.5ul Total 100ul 400ul
94C 3min -> 11 cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
Bioneer column purification. Yield: No_1: 30.5ng/ul x 30ul No_9: 44.7ng/ul x 30ul
Digestion with MmeI[edit]
No_1_9 x2 Total 30ul dUTP_PCR 20ul 10X NEBuffer 4 4ul 8 1mM SAM(fresh) 4ul 8 2U/ul Mme I 8ul 16 ddH2O 4ul 8 1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h. MinEulte column purification. Elute in 12ul EB.
File:20080912 dUTP PCR and MmeI of No 1 9.jpg20080912_dUTP_PCR and MmeI of No_1_9
step3. USER digestion[edit]
DNA 10ul USER 3ul total 13ul
37C 12h.
step4. S1 nuclease digestion[edit]
No1_9 x2 10 x S1 nuclease buffer: 2ul 4ul DNA after USER digestion: 13ul 13ul S1 nuclease (10U/ul): 1ul 2ul ddH2O 4ul 8ul
37C 10mins. Minelute cloumn purify. Elute in 16ul H2O.
step5. end repair[edit]
No1_9 positive control x3 Total 25ul H2O 13ul DNA 15ul 2ul(20080801_spacer_107bp) dNTP 2.5ul 2.5ul 7.5 dATP 2.5ul 2.5ul 7.5 10xendrepair buffer 2.5ul 2.5ul 7.5 enzyme 0.5ul 0.5ul 1.5 extra ATP(10mM) 2.5ul 2.5ul 7.5
Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 28ul H2O. Take 14ul to do the adapter ligation.
step6. adapter ligation[edit]
adaptor constraction: add 10ul Solexa_2_PE_up and 10ul Solexa_2_PE_lo_noP. 95C 5mins -> 65C 30mins -> 4C. No1_9 positive control negative control x4 DNA 13ul 13ul ddH2O 13ul 100uM Solexa_1 adaptor 0.5ul 0.5ul 0.5ul 2 100uM Solexa_2_PE adaptor 0.5ul 0.5ul 0.5ul 2 2xQuickLiage buffer 15ul 15ul 15ul 60 QuickLigase enzyme(NEB) 1ul 1ul 1ul 4 extra ATP(10mM) 2.5ul 2.5ul 2.5ul 10
Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul H2O. TBE gel size selection of ~175bp fragment. Elute in 15ul ddH2O.
File:20080913 gel cut after adaptor ligation.jpg20080913_gel cut after adaptor ligation
step7. Nick-translation[edit]
set up the ligation system: x4 Total 20ul DNA 15ul 10x ThermoPol buffer 2ul 8ul 10mM dNTP 0.4ul 1.6ul 1mg/ml BSA 2ul 8ul Bst polymerase(8U/ul) 1ul 4ul
65C for 25 minutes -> keep on ice.
step8. PCR of sequencig library[edit]
x4 Nick-translated DNA 20ul Solexa_PCR_up(10uM) 2ul 8ul Solexa_PCR_lo(10uM) 2ul 8ul 2xiProof master mix 50ul 200ul 50x SYBG 0.8ul 3.2ul ddH2O 25.2ul 100.8ul 98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
File:20080913 parellel sequencing library of No 1 9.jpg20080913_parellel sequencing library of No_1_9
Qiaquick column purification. Yields:
No_1(BJ): 17.2ng/ul (104nM) x 30ul No_9(Hybrid_2): 18.6ng/ul (112nM) x 30ul
Send 15ul of each sample to Bing Ren on 09/15/08.
Blunt cloning of sequencing library and sequencing (2008-10-16)[edit]
refer to LabNotes on [2]