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

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.pngToday's project is... Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

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.