Alice:LabNotes/2010-4-23: Difference between revisions

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                                                                       x4 well each
                                                                       x4 well each
  DNA (0.353ng/ul)          2.5ul
  DNA
  Phusion HF PCR Master Mix (2x): 50ul
  Phusion HF PCR Master Mix (2x): 50ul
  PCR_R (100uM):                    0.2ul
  PCR_R (100uM):                    0.2ul

Revision as of 18:08, 23 April 2010

PCR re-amplification of the sequencing libraries with PAIR-END primer

  • samples to be re-amplified: CViB, DF, foreskin, ips17A, ips17B, ips11B, Hfib 11
                                                                     x4 well each
DNA
Phusion HF PCR Master Mix (2x): 	50ul
PCR_R (100uM):                    0.2ul
PCR_F (100uM):                     0.2ul
SYBR Green 50x:                  0.4ul 
H2O:                         48ul                
Total:                     	100ul
98C 30sec -> (98C 10sec -> 55C 45sec -> 72C 30sec) x2 cycles -> (98C 10 sec -> 72C 30sec) x 3cycles -> 72C 5min -> hold at 4C

Sanger validation of candidate mutations

DNA preparation

  • prepared the following stock solutions for PCR templates
    Sample     Conc.     Volume      H2O       Final Conc.  
    PGP1F      
    PGP1-ips

PCR

  • 6 reactions total.
                                     x3           x2
   DNA(2ng/ul)              3ul     15ul
   Taq 2x master mix        25ul    125ul
   10uM  F primer            1ul     -
   10uM  R primer            1ul     -
   H2O                      20ul    100ul
   total                    50ul    250ul

94C 2min -> 35x (94C 30s -> 57C 30s -> 72C 30s) -> 72C 3min.
  • use 2ul to run a TBE gel to check the PCR results, then save 18ul for backup, and use the rest to do Qiaquick column purification elute in 30ul EB.
  • Use Nanodrop to check the concentration of the purified PCR product.
  • TBE gel result:
  • Prepare the samples for sequencing:
  • USE Pre-Mixed Samples Preparation Guidelines from Genewiz to mix the DNA and custom primers.
  • The DNA size is 200-500bp, so we use 10ng of DNA in 10ul volume (~1ng/ul)
  • Add 1ul of purified PCR product with 9ul of ddH2O.
  • Add the Forward primer for sequencing, dilute the Forward primer to 5uM, add 5ul of primer(total of 25pmol) and get a final volume of 15