Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/11/03
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Performing Ligase Cycling Reaction (LCR) today. See instructions here.
Prep Bridge Oligos
Make 300 nM working solutions of the dsDNA oligo bridges:
Prep DNA fragments
Dilute purified DNA from Phusion PCR to 30 nM:
Since HPK-CFP fragment is at too low a concentration, will use it straight instead.
Use polynucleotide kinase (PNK) to add 5' phosphate groups to DNA fragments.
Ligase Cycling Reaction
In a PCR tube, set up the following reaction. Use 3 µL of each dsDNA oligo bridge (9 µL total).
Thermal cycler program: