Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/29
|Plasmid Prep & Validation of DBN004_pSB1A3||Main project page|
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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.
All twelve colonies grew in liquid culture. Samples were prepared using the Sigma GenElute Plasmid Miniprep kit.
Digest for 10 minutes at 37°C.
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:
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: