Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2016/01/06
|Today's project is...|| Main project page|
Previous entry Next entry
Cloning HPK-CFP part (from KAH184) in front of EM7:ZeoR in v0120 vector. Also performing PCR on HA2 fragment to add BioBrick restriction sites.
KAH184 plasmid is at 220 ng/µL. EM7:ZeoR_v0120 plasmid is at 629 ng/µL.
Incubate at 37 °C for 10 minutes.
ladder - CFP - zeoR
HPK-CFP part: take the ~2200bp fragment.
EM7-ZeoR part: large fragment (~3600bp).
EM7:ZeoR_v0120 - 18 ng/µL
HPK:CFP - 17 ng/µL
NOTE: 260/280 for HPK:CFP part was measured at 4. Ran a spectrophotometric scan on the sample and there's no peak at 260 or 280 - probably very little DNA present in the sample. If transformation fails, this is most likely culprit.
Incubate at RT for 10 minutes.
Follow the traditional transformation protocol found here.
Homology Arm 2 PCR
Prepare master mix:
PCR extension times: