Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/11/09
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OverviewRepeating LCR, this time using the HPK-CFP fraction before gel purification. Using the steps outlined here and here. MethodsPrep Bridge Oligos Done previously Prep DNA fragments Dilute purified DNA from Phusion PCR to 30 nM:
All other DNA fragments prepped last week. PNK Reaction 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:
PCR on LCR fragment Reaction volume: 75 µL (split into 3 tubes to be pooled later) Going to try using Taq polymerase because of its 5'-->3' exonuclease activity to cleave oligo bridges. Hypothesize that Phusion's lack of strand displacement or exonuclease activity is what caused previous PCR to fail.
Reactions:
All primers have annealing temperature at 60°C Thermal cycler program:
ResultsGel image: From left to right: 1kb+ ladder, pMaxGFP, LCR (unamplified), PCR of LCR (3 lanes) Looks like I have product! Expected size is ~2500bp, and there's bands a little bit above the 2000bp band in the ladder. Weak though. Primers seem to have a stronger affinity for something else... primer dimerization going on perhaps? Or the dye in the GoTaq green master mix is fluorescing right there. That seems likely, actually. Will have to try again, using GoTaq clear master mix, and give myself time to do gel extraction. |