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=PCR re-amplification of the sequencing libraries with PAIR-END primer= *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== [[File:ZhangLab_2 2010-04-25 16hr 20min.jpg]] ==PAGE quantification== [[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= ==DNA preparation== * 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== * 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== [[File:mutations.jpg | 700px]]
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