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:

2015-11-11 E-S digestion DBN001 pSB1A3 DBN008.JPG

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.