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

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Overview Repeating GoTaq PCR on LCR fragment, cleaning up, and then digesting and inserting into a vector for transformation. Methods GoTaq PCR on LCR I'll be using 320 µL total reaction volume, split into 16 PCR tubes (20 µL each) Reagent Stock Volume used (µL) GoTaq 2x MM 2x 160 Primer (for) 100 µM 4 Primer (rev) 100 µM 4 Template 50 ng/µL 4 H2O - 148 Total 320 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 15 seconds 60°C 30 seconds 68°C 150 seconds Final Extension 68°C 5 minutes Hold 4°C infinite Digestion with EcoRI/SpeI Following instructions here. Stopped before transformation, after gel extraction (see below). Results Gel: 1kb+ ladder, vector (pSB1A3), insert. Expected vector size after treatment with E/S is ~2000bp. Expected insert size is ~2500bp. Something's going wrong with the PCR product - either the LCR made lots of a small fragment (possible, compare to un-amplified LCR product here, or the PCR is amplifying the wrong size fragment. Also, the large fragment in the product looks to be about 500bp too small. Will try a transformation tomorrow, but not hopeful.