Jie:LabNotes/CpgSeq/2008-9-12: Difference between revisions
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>Jie deng (New page: ==sequencing library construction for paralleling sequencing == Use the captured PCR mix of No_1(BJ) and No_9(Hybrid_2) as the templates. ==2nd PCR amplification with AmpF6.3/AmpR6.3 an...) |
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==sequencing library construction for paralleling sequencing == | ==sequencing library construction for paralleling sequencing == | ||
Use the captured PCR mix of No_1(BJ) and No_9(Hybrid_2) as the templates. | Use the captured PCR mix of No_1 (BJ) and No_9 (Hybrid_2) as the templates. refer to LabNotes on [http://genome-tech.ucsd.edu/LabNotes/index.php/Jie:LabNotes/CpgSeq/2008-8-15] | ||
==2nd PCR amplification with AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40 == | ==2nd PCR amplification with AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40 == |
Revision as of 16:28, 12 September 2008
sequencing library construction for paralleling sequencing
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
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) 0.5ul 2ul Total 100ul 400ul
94C 3min -> 9cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
Bioneer column purification. Yield: No_1: 33.2ng/ul x 30ul No_9: 49.4ng/ul x 30ul
Digestion with MmeI
No_1_2_3_9 x4 Total 30ul dUTP_PCR 20ul 10X NEBuffer 4 4ul 16 1mM SAM(fresh) 4ul 16 2U/ul Mme I 8ul 32 ddH2O 4ul 16 1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h. MinEulte column purification. Elute in 12ul EB.
step3. USER digestion
DNA 10ul USER 3ul total 13ul
37C 12h.
step4. S1 nuclease digestion
No1_2_3_9 x4 10 x S1 nuclease buffer: 2ul 8ul DNA after USER digestion: 13ul 13ul S1 nuclease (10U/ul): 1ul 4ul ddH2O 4ul 16ul
37C 10mins. Minelute cloumn purify. Elute in 16ul H2O.
step5. end repair
No1_2_3_9 positive control x5 Total 25ul H2O 13ul DNA 15ul 2ul(20080801_spacer_107bp) dNTP 2.5ul 2.5ul 12.5 dATP 2.5ul 2.5ul 12.5 10xendrepair buffer 2.5ul 2.5ul 12.5 enzyme 0.5ul 0.5ul 2.5 extra ATP(10mM) 2.5ul 2.5ul 12.5
Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.
step6. adapter ligation
No1_2_3_9 positive control negative control x6 DNA 13ul 13ul ddH2O 13ul 48 100uM Solexa_1 adaptor 0.5ul 0.5ul 0.5ul 3 100uM Solexa_2 adaptor 0.5ul 0.5ul 0.5ul 3 2xQuickLiage buffer 15ul 15ul 15ul 90 QuickLigase enzyme(NEB) 1ul 1ul 1ul 6 extra ATP(10mM) 2.5ul 2.5ul 2.5ul 15
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.
step7. Nick-translation
set up the ligation system: x6 Total 20ul DNA 15ul 10x ThermoPol buffer 2ul 12ul 10mM dNTP 0.4ul 2.4ul 1mg/ml BSA 2ul 12ul Bst polymerase(8U/ul) 1ul 6ul
65C for 25 minutes -> keep on ice.
step8. PCR of sequencig library
x6 Nick-translated DNA 20ul Solexa_PCR_up(10uM) 2ul 12ul Solexa_PCR_lo(10uM) 2ul 12ul 2xiProof master mix 50ul 300ul 50x SYBG 0.8ul 4.8ul ddH2O 25.2ul 151.2ul 98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.