Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/11/11
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Repeating GoTaq PCR on LCR fragment, cleaning up, and then digesting and inserting into a vector for transformation.
GoTaq PCR on LCR
I'll be using 320 µL total reaction volume, split into 16 PCR tubes (20 µL each)
All primers have annealing temperature at 60°C
Thermal cycler program:
Digestion with EcoRI/SpeI
Following instructions here.
Stopped before transformation, after gel extraction (see below).
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.