Alice:LabNotes/2009-9-21: Difference between revisions

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>Zsakura2
(New page: *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== Vaccum...)
 
>Sam Chiang
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==single-end Shotgun sequencing library construction==
==single-end Shotgun sequencing library construction==


  VaccumFuge the sheared DNA to ~38ul (use setting 45C for ~1hr)
  VaccumFuge the sheared DNA to ~38ul (use setting 60C for ~30min)


===End repair (epicentre)===
===End repair (epicentre)===
Line 31: Line 31:


  - The adapter:DNA molar ratio is at least 20:1
  - The adapter:DNA molar ratio is at least 20:1
  - Purification with Qiagen MinElute (elute with 16 ul EB buffer)
  - Purification with Qiagen MinElute (elute with 12 ul EB buffer)


===Nick repair===
===Nick repair===
(split into two batch)
  DNA from above 10
  DNA from above 8
  10x ThermoPol buffer (NEB) 1.2
  10x ThermoPol buffer (NEB) 1.2
  10 mM dNTP 0.5
  10 mM dNTP 0.5
Line 42: Line 41:


===PCR amplification===
===PCR amplification===
  DNA from above 10
  DNA from above 6
  PCR_up (100uM) 0.2
  PCR_up (100uM) 0.2
  PCR_lo (100uM) 0.2
  PCR_lo (100uM) 0.2
  2x Phusion GC master mix         50
  2x Phusion GC master mix       50
  50x SYBR Green I (Invitrogen) 0.2
  50x SYBR Green I (Invitrogen) 0.2
  dH2O 40
  dH2O 40
  98C 1min -> 5 cycles of (98C 10sec -> 64C 20 sec -> 72C 20sec) -> 72C 5min
  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)
  - Use real time machine, the number of cycles x is determined by real time curve (stop the rxn before reaching plateau)
Line 62: Line 61:
  Syb_FP5(100uM) 0.2
  Syb_FP5(100uM) 0.2
  Syb_RP7(100uM) 0.2
  Syb_RP7(100uM) 0.2
  2x Phusion GC master mix        50
  2x Phusion GC master mix        25
  50x SYBR Green I (Invitrogen) 0.2
  50x SYBR Green I (Invitrogen) 0.2
  dH2O                   40
  dH2O                   24
  split every 100ul rxn into 2 wells, so total rxn vol for PCR setup is 50ul
  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
  98C 30sec -> 13-20 cycles of (98C 10sec -> 64C 20 sec -> 72C 20sec) -> 72C 5min

Revision as of 17:55, 24 September 2009

  • 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

VaccumFuge the sheared DNA to ~38ul (use setting 60C for ~30min)

End repair (epicentre)

  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

- 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

Ligation with adapters

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

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

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

- The size range of the library DNA is ~175-225 bp.


QPCR

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