Alice:LabNotes/2010-4-23

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

  • samples to be re-amplified:
    • CViB: 5.27nM =10.43ng/ul
    • DF: 81.7nM = 161.77ng/ul
    • foreskin: 75.38nM = 149.25ng/ul
    • ips17A: 2.04nM = 4.04ng/ul
    • ips17B: 2.01nM = 3.9ng/ul
    • ips11B: 3.81nM = 7.54ng/ul
    • Hfib 11: 3.22nM = 6.38ng/ul
    • CV1: 1.34nM= 3.1ng/ul (done on 4/26/2010)
                                          x4 well each
DNA                    1ul
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
  • Qiaquick column purification and elute in 50ul EB.
  • nanodrop result:
  • CViB: 32.4ng/ul
  • DF: 29.5ng/ul
  • Hfib 11: 19.1ng/ul
  • 11b: 24.2ng/ul
  • 17b: 15.5ng/ul
  • 17a: 17.6ng/ul
  • FS: 33.9ng/ul
  • CV1: 23.1ng/ul (done on 4/26/2010)
  • AMPure beads purification using 1x total reaction volume (50ul beads) and elute in 40ul ddH2O

result[edit]

File:ZhangLab 2 2010-04-25 16hr 20min.jpg

PAGE quantification[edit]

File:ZhangLab 2 2010-04-27 13hr 37min.jpg

  • 37.16nM= hFib11 (350bp)
  • 57.73nM= ips11b (350bp)
  • 61.86nM= CviB (350bp)
  • 26.90nM= ips17b (350bp)
  • 74.71nM= FS (300bp)
  • 32.37nM= ips17a (350bp)
  • 67.09nM= DF (300bp)
  • 51.07nM= CV1 (350bp)
  • 84.67nM= CviF (350bp)

Sanger validation of candidate mutations[edit]

DNA preparation[edit]

  • prepared the following stock solutions for PCR templates
    Sample     Conc.      Volume      H2O       Final Conc.  
    PGP1F      41.8ng/ul   2ul       39.8ul      2ng/ul
    PGP1-ips   399ng/ul    1ul       198.5ul     2ng/ul

PCR[edit]

  • 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

result[edit]

File:Mutations.jpg