Kun:LabNotes/ExonomeSeq/2009-5-23
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Shotgun library construction
- Start with four normalized and size-selected libraries captured from PGP1-3, NA12878 (prepared by Alice Li). The estimated concentration is ~2ng/ul each.
PCR with dUTP
x 4 x 4 Template: 1ul 2x EconoTaq Master Mix 50ul 800 100uM AmpF6.3NH2 0.2ul 3.2 100uM AmpR6.3NH2 0.2ul 3.2 10mM dUTP 0.6ul 9.6 50x SYBG I 0.4ul 6.4 H2O 48ul 770 94C 3min -> 9 cycles of (94C 30sec -> 58C 30sec -> 72C 20sec) -> 72C 3min -> 4C Purified every 4x50ul for each amplicon with two MinElute column. A: 23ng/ul x 20ul B: 14ng/ul x 20ul C: 14ng/ul x 20ul D: 18ng/ul x 20ul
Purified each of the remaining amplicons with one DNA concentrator-5 column, eluted with 20ul EB A: 6.3ng/ul x 20ul B: 6.2ng/ul x 20ul C: 6.8ng/ul x 20ul D: 4.8ng/ul x 20ul
USER digestion
DNA 20ul USER enzyme 2ul 37C 1h
S1 nuclease digestion
10 x S1 nuclease buffer: 3ul DNA after USER digestion: 22ul S1 nuclease (10U/ul): 2ul ddH2O 2ul
37C 15mins. Purified with MinElute cloumn. Elute in 16ul H2O.
End repair (Enzymatics End-repair Mix)
Fragmented DNA 15ul dNTP 2.5ul 10x buffer 2.5ul Enzyme mix 1ul H2O 4ul Incubate at RT for 30 minutes Purified with Qiaquick column. Select the ~100bp fragments with 2% SizeSelect gel
Adaptor ligation(Enzymatic T4 ligase vs. NEB QuickLigase)
NEB DNA 20ul 2X Rapid Ligation buffer 25ul 100uM Solexa_1 adaptor 0.5ul 100uM Solexa_2 adaptor 0.5ul T4 DNA ligase(600U/ul) 1ul H2O 3ul
Keep at room temperature (~25C) for 10 minutes. Purify with MinElute columns.
Nick translation
DNA 8ul 10x ThermoPol buffer 1ul 10mM dNTP 0.4ul Bst Pol (8U/ul) 0.5ul 65C 10min
PCR
x2 DNA 10ul Solexa_PCR_up(10uM) 2ul Solexa_PCR_lo(10uM) 2ul 2x iProof master mix 50ul 50x SYBGI 0.4ul ddH2O 36ul 98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) 5 cycles of (98C 10sec -> 72c 20sec) -> 72C 3min -> 4C hold.