Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/12/11

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Overview Repeating PCR of pSB1A3_BsmBI fragment. Reagent Stock Volume (µL) GC Buffer 5x 10 dNTP mix 10 mM 1 DMSO - 1.5 Phusion 20 U/µL 0.5 Template 50 ng 0.3 Primer (for) 100 µM 0.2 Primer (rev) 100 µM 0.2 H2O - 36.3 Total 50 Template: DBN001_pSB1A3 (close enough) Primers: P140, P141 PCR protocol: basic Phusion, 60°C annealing temp, 90 second extension time, 35 cycles. Results No amplification, because no binding site for reverse primer on selected template. Picked up some I13422_pSB1A3 from Wang Lab (pSB1A3 vector with pTet & GFP insert), that should work better. Will repeat attempt on Monday.