Alice:LabNotes/2010-1-22
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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.
- 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 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