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