Alice:LabNotes/2009-8-26
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single-end Shotgun sequencing library construction[edit]
capture reaction is under LabNotes 8/15/09 and 8/16/09; MmeI preparation is under LabNotes 8/20/09
received 7 tubes from Billy VaccumFuge the sheared DNA to ~38ul (use setting 45C for ~1hr)
End repair (epicentre)[edit]
Fragmented DNA 38ul 10x buffer 5ul 2.5mM dNTP 5ul 10mM ATP 5ul enzyme 1ul (mix everything before adding enzyme)
Incubate at RT for 45 min
PAGE gel size selection and purification[edit]
- Minimize the number of samples per gel to avoid cross contamination (ideally 1 sample/gel) - Do not overload the gel (max 500ng/lane for Invitrogen 10-well gel) - Cut the size range at ~80-130 bp - PAGE gel purification, final elute: 220 uL File:ZhangLab 2 2009-08-27 14hr 53min-gel cut.jpg File:ZhangLab 2 2009-08-27 16hr 17min.jpg File:ZhangLab 2 2009-08-27 16hr 15min.jpg File:ZhangLab 2 2009-08-27 14hr 51min-gel cut.jpg
Ligation with adapters[edit]
DNA from above 22 2x Enzymatics Quick Ligase buffer 25 Adapter 1 (100 uM) 1 Adapter 2 (100 uM) 1 Enzymatics Quick Ligase 1 (mix everything before adding enzyme) Room temp 15 minutes
- The adapter:DNA molar ratio is at least 20:1 - Purification with Qiagen MinElute (elute with 16 ul EB buffer)
Nick repair[edit]
(split into two batch) DNA from above 8 10x ThermoPol buffer (NEB) 1.2 10 mM dNTP 0.5 BST (8 u/ul) (NEB) 0.5 65C 10 minutes
PCR amplification[edit]
DNA from above 10 PCR_up (100uM) 0.2 PCR_lo (100uM) 0.2 2x Phusion GC master mix 50 50x SYBR Green I (Invitrogen) 0.2 dH2O 40 98C 1min -> 5 cycles of (98C 10sec -> 64C 20 sec -> 72C 20sec) -> 72C 5min
- Use real time machine, the number of cycles x is determined by real time curve (stop the rxn before reaching plateau) - Check the amplicon size and concentration with 6% PAGE gel
Size selection and purification of the constructed sequencing libraries[edit]
- The size range of the library DNA is ~175-225 bp.
TBE gel image before gel cut: File:ZhangLab 2 2009-08-28 11hr 38min.jpg TBE gel image after gel cut: File:ZhangLab 2 2009-08-28 16hr 48min.jpg
Gel quantification result: DF6-9-9 60nM foreskin 45nM PGP1-ips 38nM CV-ips 90nM CV-fibroblast 76nM PGP1 34nM PGP2 34nM
QPCR[edit]
DNA 1 Syb_FP5(100uM) 0.2 Syb_RP7(100uM) 0.2 2x Phusion GC master mix 50 50x SYBR Green I (Invitrogen) 0.2 dH2O 40 split every 100ul rxn into 2 wells, so total rxn vol for PCR setup is 50ul 98C 30sec -> 13-20 cycles of (98C 10sec -> 64C 20 sec -> 72C 20sec) -> 72C 5min File:ZhangLab 2 2009-08-31 10hr 30min.jpg