Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/05/07
|DBN006 PCR verification & gel purification||Main project page|
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Yesterday I performed PCR on the DBN006 insert (from colonies 1 and 3) to try and add Type IIS restriction sites to the insert. Today I'll be checking the PCR product and performing a gel purification.
1kb+ ladder, colony 1, colony 3
The expected fragment size (~2kb) is present, but so is a large smear. Going to move forward with gel purification.
Bands were excised and weighed.
Colony 1 gel mass: 0.026g
Colony 3 gel mass: 0.038g
Samples were prepped using the Sigma GenElute Gel Extraction Kit, and concentrated into a final volume of 25 µL.
Single-Step Digestion/Ligation Reaction
NOTE: Steps performed are from the Zhang lab's CRISPR cloning protocol, following only step 2 of the single-step digestion-ligation reaction. Volumes for inserts are calculated from their concentrations post-PCR cleanup.
Incubate the ligation reaction in a thermocycler:
Transform with 10µL into DH5-α Turbo competent E. coli. Use a negative plate with 10µL M.B. grade H2O as a control, for 4 plates total.