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