Alice:LabNotes/2009-6-4
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Shotgun library construction
PCR with dUTP
x 4 x4 Template: 0.25ul 1ul 2x EconoTaq Master Mix 50ul 200ul 100uM AmpF6.3NH2 0.2ul 0.8ul 100uM AmpR6.3NH2 0.2ul 0.8ul 1mM dUTP 4ul 16ul 50x SYBG I 0.4ul 1.6ul H2O 50ul 770ul 94C 3min -> 10 cycles of (94C 30sec -> 58C 30sec -> 72C 20sec) -> 72C 3min -> 4C Purified the amplicon with one Qiaquick column each e-1: 14.2ng/ul x 30ul e-2: 31.1ng/ul x 30ul f-1: 13.3ng/ul x 30ul f-2: 33.6ng/ul x 30ul
USER digestion
Qiaquick purified DNA 15ul USER enzyme 2ul 37C 1h
S1 nuclease digestion
10 x S1 nuclease buffer: 2ul DNA after USER digestion: 17ul S1 nuclease (10U/ul): 2ul ddH2O 2ul
37C 15mins Purified with MinElute cloumn. Elute in 18ul H2O.
End repair (epicentre)
Fragmented DNA 15ul dNTP 2.5ul 10x buffer 2.5ul enzyme 0.5ul ATP 5ul Incubate at RT for 40 minutes Purified with minelute column and elute in 20ul Select the ~100bp fragments with TBE gel
Adaptor ligation
DNA 20ul 2X Rapid Ligation buffer 27ul 100uM Solexa_1 adaptor 1ul 100uM Solexa_2 adaptor 1ul QuickLigase(400U/ul) 2ul Keep at room temperature (~25C) for 10 minutes. Purify with MinElute columns and elute in 16ul EB.
performed one round of size selection on positive control tube using 2% Size Select E-gel. The total amount obtained is 25ul.
Nick translation
DNA 15ul 10x ThermoPol buffer 2ul (3ul for positive control) 10mM dNTP 0.4ul BSA 2ul Bst Pol (8U/ul) 1ul 65C 20min
PCR
x 5 DNA 10ul Solexa_PCR_up(100uM) 0.2ul Solexa_PCR_lo(100uM) 0.2ul 2x iProof master mix 50ul 50x SYBGI 0.4ul ddH2O 40ul 98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 15sec) 6 cycles of (98C 10sec -> 72c 20sec) -> 72C 3min -> 4C hold.
x4 DNA 10ul (second half of the products) Solexa_PCR_up(100uM) 0.2ul Solexa_PCR_lo(100uM) 0.2ul 2x iProof master mix 50ul 50x SYBGI 0.4ul ddH2O 40ul 98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 15sec) 5 cycles of (98C 10sec -> 72c 20sec) -> 72C 3min -> 4C hold.
Purify with one qiaquick column for each set, and elute in 40ul EB a: 142.9ng/ul b: 101.2ng/ul c: 117.2ng/ul d: 126.5ng/ul
perform gel size selection at 150-160bp
Ethanol precipitation on the sets from above, and also four tubes obtained from Dr. Zhang. Elute in 40ul of ddH2O each.