Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/11/04

Today's project is...

Main project page Previous entry      Next entry

Overview Phusion PCR on LCR fragment. Reaction volume: 100 µL (split into 4 tubes to be pooled later) Reagent Stock Volume used (µL) GC Buffer 5x 20 dNTP mix 10 mM 2 Primer (for) 100 µM 1 Primer (rev) 100 µM 1 Template 50 ng/µL 15 DMSO - 3 Phusion 20/µL 1 H2O - 57 Total 100 Reactions: Reaction Forward primer Reverse primer template DBN008 P40 P41 LCR product All primers have annealing temperature at 60°C Thermal cycler program: STEP TEMP TIME Initial Denaturation 95°C 30 seconds 30 Cycles 95°C 10 seconds 60°C 20 seconds 72°C 60 seconds Final Extension 72°C 10 minutes Hold 4°C infinite Results Nonspecific binding and amplification in the sample. Not sure what's going wrong here... this is after PCR of the LCR product, using forward primer for HA1 and reverse primer for HA2. Product size should be approximately 2600-2700bp