Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/05/12

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Overview

Taking the liquid cultures started yesterday and performing a plasmid miniprep on them, followed by DNA quantification, restriction digest, and gel electrophoresis to validate.

Plasmid Miniprep

Kit used: Sigma GenElute Plasmid Miniprep

DNA Quantification

Sample ID Conc. (ng/µL) St Dev (ng/µL) 260/280
DBN007 1.1 365.0 57.81 1.91
DBN007 1.2 306.8 10.66 1.94
DBN007 1.3 329.0 17.55 1.94
DBN007 3.1 342.8 17.36 1.92
DBN007 3.2 311.7 9.23 1.92
DBN007 3.3 358.8 7.39 1.92

Restriction Digest

Reagent Volume (µL)
Water 23
DNA 2
10x FD Buffer (Green) 3
ApaLI 1
SpeI 1
Total 30

Incubate at 37°C for 10 minutes.

Gel Electrophoresis

Expected size fragments are 2.5kb, 1.2kb, and 500bp.

2015-05-12 DBN007 pSB1A3 ApaLI SpeI digestion annotated.png

None of the colonies contain the insert. Debating whether to try again with new colonies or try another approach... other option would be to PCR-amplify the two homology arms, adding restriction enzyme cut sites, then doing standard biobrick cloning to place the homology arms and DBN006 fragment into v0120 plasmid.