Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/03/23

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Overview

Today I'll be performing another PCR, this time using the correct template (KAH57) and the DBN002 primers. To save on template I'll be using 20μL reaction volume instead of 50μL, so the reagent volumes will look like this:

Reagent Volume (uL) for 9 rxns (uL)
2x GoTaq MM 10 90
MB grade H2O 9.6 86.4
forward primer 0.1 0.9
reverse primer 0.1 0.9
template 0.2 1.8
Total 20 180

I'll be running a thermal gradient again from 52°C to 59°C, for 8 reactions in total (+1 for pipetting error).

Gel Electrophoresis

Gel layout:

1kb+ 52C 53C 54C 55C 56C 57C 58C 59C

Lanes were loaded with 2μL PCR product, 8μL H2O and 1μL 10x loading dye.

Gel image:

2015-03-23 PCR products annotated.png

Two bands result from the two possible binding sites of the forward primer. The ~1500bp band is the one we want, as that contains both copies of the AmCyan gene. Unfortunately it's also the fainter of the two bands; I'll have to do some gel purification on the PCR products. Reactions run at higher annealing temperatures (59°C) appear to have slightly higher yield of the amplification product of interest.