Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/27
|Plasmid Verification of DBN001_pSB1A3 & Type IIS Digestion/Ligation of DBN004_pSB1A3||<html><img src="/images/9/94/Report.png" border="0" /></html> Main project page|
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I'll be performing a restriction digest on the DNA harvested from the DBN001_pSB1A3 ligation & transformation, then running the digestion products on a gel to check size fragments. Hopefully I'll see size fragments that correspond to a successful ligation.
Digest for 10 minutes at 37°C.
Expected size fragments: 2132bp (backbone), 224bp (insert)
Colonies 1, 2, and 5 have bands of the expected sizes. Going to go ahead and perform Type IIS Restriction Digest/Ligation.
DBN003 Post-PCR DNA Quantification
Better late than never... finally quantifying the amount of PCR product from amplifying the DBN003 fragment (CPK & Kozak).
Proceeding to PCR clean-up using the QIAquick PCR purification kit.
DNA concentration after using the Qiagen kit:
Phosphorylate & Anneal Oligo Pairs
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.