Alice:LabNotes/2009-1-13: Difference between revisions
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>Jie deng |
>Jie deng |
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==2nd PCR amplification== | ==2nd PCR amplification== | ||
AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40 | AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40 | ||
system setup: | system setup: x4 | ||
H2O 43.2ul 172.8ul | H2O 43.2ul 172.8ul | ||
2x Master mix 50ul 200ul | 2x Master mix 50ul 200ul | ||
dUTP(1mM) 2ul 8ul | dUTP(1mM) 2ul 8ul | ||
AmpF6.3(10uM) 2ul 8ul | AmpF6.3(10uM) 2ul 8ul | ||
AmpR6.3(10uM) 2ul 8ul | AmpR6.3(10uM) 2ul 8ul | ||
50x SYBG I 0.4ul 1.6ul | 50x SYBG I 0.4ul 1.6ul | ||
template( | template(19ng/ul) 0.1ul 0.4ul | ||
Total 100ul 400ul | Total 100ul 400ul | ||
94C 3min -> | 94C 3min -> 9 cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C | ||
Bioneer column purification. | Bioneer column purification. | ||
==step2. Digestion with MmeI== | ==step2. Digestion with MmeI== |
Revision as of 21:19, 13 January 2009
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 Bioneer column purification.
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 22ul EB.
step3. USER digestion
x2 DNA 10ul 20ul USER 3ul 6ul 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
positive control Total 25ul H2O 13ul DNA 15ul 2ul(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
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.