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 30ul USER enzyme 2ul 37C 1h
S1 nuclease digestion
10 x S1 nuclease buffer: 4ul DNA after USER digestion: 32ul S1 nuclease (10U/ul): 1ul ddH2O 3ul
37C 15mins Purified with MinElute column. Elute in 18ul H2O.
Nanodrop: e-1:5ng/ul e-2:7.1ng/ul f-1:6.8ng/ul f-2:10.1ng/ul
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 Egel.
Adaptor ligation
pos. control neg control DNA 16ul 20ul H2O 16ul 2X Rapid Ligation buffer 18ul 22ul 18ul 100uM Solexa_1 adaptor 0.5ul 0.5ul 0.5ul 100uM Solexa_2 adaptor 0.5ul 0.5ul 0.5ul QuickLigase(400U/ul) 1ul 1ul 1ul 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 16ul.
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.
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.