Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/03/20

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Overview

Performed PCR this morning using KAH45 template and the DBN002 forward and reverse primers. Eight samples were run with annealing temperatures ranging from 51°C to 58°C. Procedure was otherwise identical to PCR run on Wednesday (see details here).

Gel Electrophoresis

Gel layout:

1kb+ Ladder51C52C53C54C55C56C57C58C

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

Gel image:

Image:2015-03-20_PCR_products.png

Expected size fragment is 1440bp, PCR results in a product about 750bp in size. This may be because the template contains two copies of the AmCyan gene and the forward primer is annealing to the second copy instead of the first.

Notes

Upon closer inspection of the template files, we should actually be amplifying KAH57, not KAH45. This is because KAH45 does not contain a STOP sequence, which is included in part of the reverse primer. So the bands I'm seeing in the gel image above probably aren't AmCyan : NLS : STOP, but something else entirely and uncharacterized.

To do next week: repeat temperature gradient, from 52°C to 59°C, using DBN002 f1 and DBN002 r1 primers and KAH57 template.


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