Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2016/01/06
Today's project is... | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
OverviewCloning 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. Restriction Digest
Incubate at 37 °C for 10 minutes. Gel Extractionladder - CFP - zeoR HPK-CFP part: take the ~2200bp fragment. EM7-ZeoR part: large fragment (~3600bp). DNA QuantificationEM7: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. Ligation
Incubate at RT for 10 minutes. TransformationFollow the traditional transformation protocol found here. Homology Arm 2 PCRPrimers:
Reaction setup:
Prepare master mix:
PCR extension times:
|