Alice:LabNotes/2009-6-10
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single-end Shotgun sequencing library construction[edit]
- capture experiment and template information can be found under LabNote 6/4/09
PCR with dUTP[edit]
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 200ul 94C 3min -> 9 cycles of (94C 30sec -> 58C 30sec -> 72C 20sec) -> 72C 3min -> 4C Purified the amplicon with one Qiaquick column each e-1: 75.2ng/ul x 30ul e-2: 52.6ng/ul x 30ul f-1: 49.6ng/ul x 30ul f-2: 62.8ng/ul x 30ul
File:ZhangLab 2 2009-06-09 12hr 01min-dutp.jpg
USER digestion[edit]
Qiaquick purified DNA 30ul USER enzyme 3ul 37C 1h
S1 nuclease digestion[edit]
10 x S1 nuclease buffer: 4ul DNA after USER digestion: 33ul S1 nuclease (10U/ul): 1ul ddH2O 2ul
37C 15mins Purified with MinElute column. Elute in 18ul H2O.
End repair (epicentre)[edit]
pos. control Fragmented DNA 18ul Spacer 107bp 3ul + H2O 12ul 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 (only the seq. library samples, positive control not included).
Adaptor ligation[edit]
pos. control DNA 16ul 20ul 2X Rapid Ligation buffer 18ul 22ul 100uM Solexa_1 adaptor 0.5ul 0.5ul 100uM Solexa_2 adaptor 0.5ul 0.5ul QuickLigase(400U/ul) 1ul 1ul ATP 3ul 3ul 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[edit]
DNA 15ul 10x ThermoPol buffer 2ul 10mM dNTP 0.4ul BSA 2ul Bst Pol (8U/ul) 1ul 65C 20min
TBE gel size selection of samples (not including control) at ~160bp
PCR[edit]
x5 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 -> 6 cycles of (98C 10sec -> 65C 20 sec -> 72C 15sec)-> 7 cycles of (98C 10 sec -> 72C 20sec) -> 72C 3min -> 4C hold. File:ZhangLab 2 2009-06-10 12hr 31min.jpg qiauqick column purification combining the two PCR tubes for one sample, and elute in 30ul of EB. nanodrop: a3: 17.3ng/ul b3: 18.2ng/ul c3: 18.4ng/ul d3: 22ng/ul
gel quantification and final concentration estimate (sent to Zhen Ye): lane 1: PGP1-3-061009: 34nM x 15ul; (4ng/ul) lane 2: PGP2-3-061009: 25nM x 15ul; (3ng/ul) lane 3: PGP3-3-061009: 22nM x 15ul; (2.6ng/ul) lane 4: NA12878-3-061009: 22nM x 15ul; (2.6ng/ul)