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

DBN007_pSB1A3 plasmid prep & validation

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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. 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.