Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/29

Plasmid Prep & Validation of DBN004_pSB1A3

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Overview After picking 12 colonies to streak and grow liquid cultures, I'm going to harvest them, perform plasmid minipreps, quantify DNA, and do a basic verification using restriction digest and gel electrophoresis. Plasmid Miniprep All twelve colonies grew in liquid culture. Samples were prepared using the Sigma GenElute Plasmid Miniprep kit. DNA Concentration Sample ID Conc. (ng/µL) St Dev (ng/µL) 260/280 1.1 108.1 2.76 1.95 1.2 140.5 11.25 1.95 1.3 161.1 1.54 1.92 1.4 146.0 2.89 1.94 2.1 149.9 0.79 1.93 2.2 185.7 24.56 1.88 2.3 193.9 21.42 1.90 2.4 110.9 1.89 1.89 5.1 149.3 8.35 1.91 5.2 148.3 0.37 1.88 5.3 142.3 17.26 1.88 5.4 123.6 1.00 1.91 Restriction Digest Reagent Volume DNA 600ng (5 µL) 10x FastDigest Buffer 3 ApaLI 1 SpeI 1 Water 20 Total 30 Digest for 10 minutes at 37°C. Gel Electrophoresis Load 15 µL of digest on the gel. Expected size fragments are 525, 1246, and 2536bp. All twelve colonies are have size fragments at ~1200, ~500 and ~400bp. This doesn't match with an SpeI/ApaLI digestion of DBN004_pSB1A3, but it does match with plain pSB1A3 when digested with the same enzymes. My guess is the few colonies that survived Type IIS digestion/ligation were the ones that could excise the insert and retain the ampR gene in the plasmid. I also didn't realize this until just now, but there's a BsaI cut site inside the AmpR gene. Should have checked that before ordering the DBN001 fragment and before amplifying the DBN002 & DBN003 fragments. Not sure how to proceed from here. Spoke with Dr. Haynes, here are the main options for improving efficiency of the Type IIS restriction/ligation: Increase ratio of insert to vector Use NEB ligase buffer instead of T4 ligase buffer In order to reduce the possible number of recombination events, I'm going to try ligating the DBN002 & DBN003 fragments together. There are a couple ways to do this, as well: Type IIS ligation using the existing fragments Traditional BioBrick cloning with conventional restriction enzymes One difficulty there: don't have primers/template for BioBrick cloning. Looking through the library doesn't show any good templates. Will have to do more searching.