Jie:LabNotes/CpgSeq/2009-1-13
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sequencing library construction (2009-01-13) of BJ, Hybrid_1, Hybrid_2, PGP1F, Hues12[edit]
No1: BJ (4.8ng/ul, 09/01/08); NO8: Hybrid_1 (4ng/ul, 08/04/08); No9: Hybrid_2 (2.8ng/ul, 09/01/08); No17: PGPF1 (7ng/ul, 10/10/08); No18: Hues12 (6ng/ul, 11/04/08).
2nd PCR amplification with AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40[edit]
reaction system x4 x5 H2O 43.2ul 172.8ul 864 2x Master mix 50ul 200ul 1000 dUTP(1mM) 2ul 8ul 40 AmpF6.3(10uM) 2ul 8ul 40 AmpR6.3(10uM) 2ul 8ul 40 50x SYBG I 0.4ul 1.6ul 8 template(18.4ng/ul) 0.1ul 0.4ul 2 Total 100ul 400ul 2000
94C 3min -> 13cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C Qiaquick column purification. Yield: BJ: 28.3ng/ul x30ul; Hybrid_1: 37.5ng/ul x 30ul; Hybrid_2: 39.1ng/ul; PGPF1: 30.7ng/ul; Hues12: 35.4ng/ul.
step2. Digestion with MmeI[edit]
x5 Total 30ul DNA 20ul 10X NEBuffer 4 4ul 20 1mM SAM(fresh) 4ul 20 2U/ul Mme I 8ul 40 ddH2O 4ul 20 1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h MinElute column purify. Elute in 11ul EB.
File:2000914 MmeI.jpg2000914_MmeI
step3. USER digestion[edit]
x5 DNA 10ul USER 3ul 15ul total 13ul
37C 12h.
step4. S1 nuclease digestion (2009-01-13)[edit]
x5 10 x S1 nuclease buffer: 2ul 10ul DNA after USER digestion: 13ul S1 nuclease (10U/ul): 1ul 5ul ddH2O 4ul 20ul
37C 10mins. Minelute cloumn purify. Elute in 16ul H2O.
step5. end repair[edit]
positive control x6 Total 25ul H2O 13ul DNA 15ul 2ul(20080801_spacer_107bp) dNTP 2.5ul 2.5ul 15 dATP 2.5ul 2.5ul 15 10xendrepair buffer 2.5ul 2.5ul 15 enzyme 0.5ul 0.5ul 3 extra ATP(10mM) 2.5ul 2.5ul 15
Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.
step6. adapter ligation[edit]
total 30ul positive control negative control x7 DNA 13ul 13ul ddH2O 13ul 100uM Solexa_1 adaptor 0.5ul 0.5ul 0.5ul 3.5 100uM Solexa_2_PE adaptor 0.5ul 0.5ul 0.5ul 3.5 2xQuickLiage buffer 15ul 15ul 15ul 105 QuickLigase enzyme(NEB) 1ul 1ul 1ul 7 extra ATP(10mM) 2.5ul 2.5ul 2.5ul 17.5
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[edit]
set up the ligation system: x7 Total 20ul DNA 15ul 10x ThermoPol buffer 2ul 14 10mM dNTP 0.4ul 2.8 1mg/ml BSA 2ul 14 Bst polymerase(8U/ul) 1ul 7
65C for 25 minutes -> keep on ice.
step8. PCR of sequencig library[edit]
x7 Nick-translated DNA 20ul Solexa_PCR_upH(10uM) 2ul 14ul Solexa_PCR_PE_loH(10uM) 2ul 14ul 2xiProof master mix 50ul 1050ul 50x SYBG 0.8ul 5.6ul ddH2O 25.2ul 176.4ul 98C 30sec -> 12 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
File:20090120 sequencing library.jpg20090120_sequencing library
Purify with Qiaquick column. Elute in 30ul EB. yield: No_1 BJ: 13.7ng/ul(75.4nM) x 30ul No_8 Hybrid_1: 15.9ng/ul(87.6nM) x 30ul No_9 Hybrid_2: 16.1ng/ul(88.7nM) x 30ul No_17 PGPF1: 19.3ng/ul(106nM) x 30ul No_18 HUES12: 13.6ng/ul(74.9nM) x 30ul