Alice:LabNotes/2009-9-21
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- detailed capture reaction can be found under labnotes 8/15/09
- detailed MmeI digestion can be found under labnotes 9/14/09
single-end Shotgun sequencing library construction[edit]
VaccumFuge the sheared DNA to ~38ul (use setting 60C for ~30min)
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 2009-09-21 13hr 51min.jpg File:ZhangLab 2 2009-09-21 13hr 49min.jpg File:ZhangLab 2 2009-09-21 14hr 51min.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 12 ul EB buffer)
Nick repair[edit]
DNA from above 10 10x ThermoPol buffer (NEB) 1.2 10 mM dNTP 0.5 BST (8 u/ul) (NEB) 0.5 65C 10 minutes
PCR amplification[edit]
split the previous products to do PCR twice DNA from above 6 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 -> 8 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.
File:ZhangLab 2 2009-09-22 11hr 48min-1.jpg File:ZhangLab 2 2009-09-22 18hr 13min.jpg
QPCR[edit]
DNA 1 Syb_FP5(100uM) 0.2 Syb_RP7(100uM) 0.2 2x Phusion GC master mix 25 50x SYBR Green I (Invitrogen) 0.2 dH2O 24 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
Result: DF6-9-9: 75nM foreskin: 80nM ips-PGP1: 80nM CV-ips: 120nM CV-fibroblast: 150nM NA12878-3: 55nM