Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/15

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Overview

Here are the colonies observed after one weekend grown at room temperature. I'll also be doing a plasmid miniprep of mCh_pSB1A3 so that I have more stocks of the cloning vector.

Plate # colonies
positive control many dozens
sample 2
negative control 2

I'm going to set up two cultures for the two colonies on the sample plate, but am not very confident of the success of the ligation given the colonies on the negative control.

mCh_pSB1A3 Plasmid Miniprep

Decent yield for all four colonies picked.

--DNA Concentration--

Sample ID Concentration (ng/µL) St Dev (ng/µL)
mCh Vector 1 392.1 15.5
mCh Vector 2 315.5 18.1
mCh Vector 3 319.7 6.3
mCh Vector 4 375.1 7.1

--Restriction Digest--

Reagent Volume
DNA 600ng (1.9 - 1.5 µL)
10x FastDigest Buffer 3
EcoRI 1
PstI 1
Water 23
Total 30

Digest for 15 minutes at 37°C.



--Gel Electrophoresis--

2015-04-15 mCherry vector digest annotated.png

Expected size fragments from EcoRI/PstI digest: 2114, 980