Kun:LabNotes/ExonomeSeq/2008-1-30
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Exp.1 Library construction on the Cosmic3760 exons from NA10835 and NA20431 (01/27/2008)[edit]
Mme I digestion on the PCR amplicon[edit]
Mix pooled 50ul amplicons from two tubes, add 1ul 32mM SAM, 4ul Mme I. 37C 2h -> 75C 20min. PAGE size selection of 50ul digestions. Split the purified DNA into two tubes, one for USER digestion and the other for SolN6 primed synthesis.
USER enzyme digestion[edit]
Add 1ul USER enzyme into 10ul of DNA in ddH2O -> 37C 10min.
End repair[edit]
Set up end repair reaction (with NEB quick blunting kit)
USER DNA 10ul 10X buffer 1.5ul 1mM dNTP 1.5ul Enzyme mix 0.5ul H2O 1.5ul RT for 30min -> 75C 10min
Ligation[edit]
DNA 15ul 50uM Solexa_1 2ul 50uM Solexa_2 2ul 2X Quick ligation buffer 20ul Quick ligase 1ul mix well -> room temperature for 20min Purified with MinElute columns.
Nick-translation[edit]
DNA 10ul 10X ThermoPol Buffer 1.2ul 10mM dNTP 0.3ul 8U/ul Bst pol 0.5ul 65C 25min -> place on ice
SolN6 primed synthesis[edit]
SolF/Rn6 DNA 10ul 10X Klenow buffer 5ul 100uM SolFn6 1ul 10mM dNTP 2ul H2O 30ul
94C 2min -> 16C 1min -> add 1ul 20U/ul Klenow exo- -> 16C 10min -> 37C 10min -> Clean up the reaction with G25 column, then add 6 ul 10X buffer, 2ul dNTp, 1ul 100uM SolRn6-> 94C 1min -> 16C 1min -> add 1ul 20U/ul Klenow exo-, 1ul T4 DNA pol-> 16C 10min -> 37C 10min -> purified with MinElute columns, eluted with 10ul EB.
PCR[edit]
Nick-translated DNA 5ul SolN6 primed DNA 5ul 2X iProof master mix 50ul 50ul 100uM Solexa_PCR_up 0.25ul 0.25ul 100uM Solexa_PCR_lo 0.25ul 0.25ul H2O 44ul 44ul 50X SYBG I 0.4ul 0.4ul 98C 30sec -> 18 cycles of (98C 10sec -> 65C 20 sec -> 68C 30sec) -> 72C 3min ->15C hold File:2008-02-01-SolLib-PCR.png I decided to do PAGE purification of the remaining 10ul PCR templates, then do PCR. File:2008-02-01-PAGE.png After shearing the gel, I directly added 100ul 1X iProof mix with 0.2X SYBR Green I but no primer. Incubate at thermal cycler at 37C for 1h, then start thermal cycling with same protocol. However, I realized after 15 cycles that I forgot to added the primers. So I took the tubes out, add Solexa_PCR_lo/up primers (0.4uM), and repeat 18 cycles of thermal cycling. I took tube A/B out, and did 5 more cycles on C/D.
PAGE purification on the A/B amplicons from PAGE purified templates, and C/D amplicons from unpurified templates. The amplification A/B didn't work. File:2008-02-01-SolLib-PCR-PAGE.png