Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2016/02/04
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Sequence for EM7:ZeoR_v0120, submitted on Monday, has come back and the QuikChange site-directed mutagenesis worked on all three colonies picked. Moving forward with restriction digests of EM7:ZeoR and HA2 parts, followed by ligation and transformation into E. coli.
Incubate at 37 °C for 10 minutes, then gel purify for insert (EM7:ZeoR_v0120, 463 bp) and vector (HA2_v0120, 3349 bp).
260/280 values are a little on the high side (~2.2-2.3). Going to proceed with ligation.
Incubate at room temperature for 10 minutes, then transform using the quick transformation method and 1 µL of ligation product. Keep the rest of the ligation product in case transformation efficiency is too low.
Will pick 4 colonies, streak and start liquid cultures, plasmid prep, then verify by restriction digest.