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

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>Zsakura2
>Zsakura2
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#Pipette 46μl of Post-Capture LM-PCR Master Mix into each of the 10 reaction tubes or wells.
#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.
#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.
98C 30sec -> (98C 10sec -> 60C 30sec -> 72C 30sec) x20 cycles -> 72C 5min -> hold at 4C
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

Revision as of 23:37, 22 January 2010

Nimble/Roche EZ Exome SeqCap preparation

  • 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

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

Step 1. Prepare the LM-PCR

  • 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. 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 
PE-POST1 Oligo, 100 μM	11μl (Final Conc.: 2μM)
PE-POST2 Oligo, 100 μM	11μl (Final Conc.: 2μM)
Total	506μl
  1. Vortex the bead-bound captured DNA to ensure homogenous mixture of beads.
  2. Pipette 46μl of Post-Capture LM-PCR Master Mix into each of the 10 reaction tubes or wells.
  3. 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.

98C 30sec -> (98C 10sec -> 60C 30sec -> 72C 30sec) x20 cycles -> 72C 5min -> hold at 4C

  1. Qiaquick column purification and elute in 50ul EB