Alice:LabNotes/2010-1-22: Difference between revisions

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==Nimble/Roche EZ Exome SeqCap preparation==
==Prepare samples for Solexa sequencing (captured with Nimblegen/Roche EZ exome kits)==
*The preparation for hybridization reaction can found under labnotes 1-10-10
*The preparation for hybridization reaction can found under labnotes 1-10-10
*The hybridization reaction setup can be found under labnotes 1-12-10
*The hybridization reaction setup can be found under labnotes 1-12-10
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====Step 1. Prepare the LM-PCR====
====Step 1. Prepare the LM-PCR====
*Note: The Post-Capture LM-PCR Master Mix and the individual PCR tubes must be prepared on ice.
*Note: The Post-Capture LM-PCR Master Mix and the individual PCR tubes must be prepared on ice.
#Prepare the Post-Capture LM-PCR Master Mix in a 1.5ml tube. The amount of each reagent needed for 10 reactions (1 captured DNA) is listed below:
#Prepare the Post-Capture LM-PCR Master Mix in a 1.5ml tube.
#Vortex the bead-bound captured DNA to ensure homogenous mixture of beads.
#Pipette 46μl of Post-Capture LM-PCR Master Mix into each of the 10 reaction tubes or wells.
#Aliquot 4μl of bead-bound captured DNA as template into 10 PCR tubes/wells. Add 4μl of PCR grade water to the negative control. Mix well by pipetting up and down 5 times.
The amount of each reagent needed for 10 reactions (1 captured DNA) is listed below:
  Phusion High-Fidelity PCR Master Mix (2x) 275μl
  Phusion High-Fidelity PCR Master Mix (2x) 275μl
  PCR grade water                            209ul  
  PCR grade water                            209ul  
  PE-POST1 Oligo, 100 μM 11μl (Final Conc.: 2μM)
  Syb_FP5, 100 μM (Final Conc.: 2μM)   11ul
  PE-POST2 Oligo, 100 μM 11μl (Final Conc.: 2μM)
  Syb_RP7, 100 μM (Final Conc.: 2μM)   11ul
  Total 506μl
  Total 506μl
#Vortex the bead-bound captured DNA to ensure homogenous mixture of beads.
98C 30sec -> (98C 10sec -> 60C 30sec -> 72C 30sec) x20 cycles -> 72C 5min -> hold at 4C
#Pipette 46μl of Post-Capture LM-PCR Master Mix into each of the 10 reaction tubes or wells.
TBE gel image result:
#Aliquot 4μl of bead-bound captured DNA as template into 10 PCR tubes/wells. Add 4μl of PCR grade water to the negative control. Mix well by pipetting up and down 5 times.
[[File:ZhangLab_2 2010-01-23 18hr 01min.jpg]]
98C 30sec -> (98C 10sec -> 60C 30sec -> 72C 30sec) x20 cycles -> 72C 5min -> hold at 4C
*Qiaquick column purification and elute in 50ul EB
#Qiaquick column purification and elute in 50ul EB

Latest revision as of 20:22, 21 March 2010

Prepare samples for Solexa sequencing (captured with Nimblegen/Roche EZ exome kits)[edit]

  • The preparation for hybridization reaction can found under labnotes 1-10-10
  • The hybridization reaction setup can be found under labnotes 1-12-10
  • The washing and Recovery of Captured DNA can be found under labnotes 1-20-10

Captured DNA Amplification Using LM-PCR[edit]

  • A total of 10 reactions are performed per sample, and subsequently combined, to minimize PCR bias.

Step 1. Prepare the LM-PCR[edit]

  • Note: The Post-Capture LM-PCR Master Mix and the individual PCR tubes must be prepared on ice.
  1. Prepare the Post-Capture LM-PCR Master Mix in a 1.5ml tube.
  2. Vortex the bead-bound captured DNA to ensure homogenous mixture of beads.
  3. Pipette 46μl of Post-Capture LM-PCR Master Mix into each of the 10 reaction tubes or wells.
  4. Aliquot 4μl of bead-bound captured DNA as template into 10 PCR tubes/wells. Add 4μl of PCR grade water to the negative control. Mix well by pipetting up and down 5 times.
The amount of each reagent needed for 10 reactions (1 captured DNA) is listed below:
Phusion High-Fidelity PCR Master Mix (2x)	275μl
PCR grade water                             209ul 
Syb_FP5, 100 μM (Final Conc.: 2μM)   11ul
Syb_RP7, 100 μM	(Final Conc.: 2μM)   11ul
Total	506μl
98C 30sec -> (98C 10sec -> 60C 30sec -> 72C 30sec) x20 cycles -> 72C 5min -> hold at 4C
TBE gel image result:
File:ZhangLab 2 2010-01-23 18hr 01min.jpg
  • Qiaquick column purification and elute in 50ul EB