Haynes Lab:Notebook/CRISPR Editing/2015/01/27

Project name

Main project page Previous entry      Next entry

01/27/2015 Picked two colonies from KAH126 plate, put them into 2mLs of LB + amp. Let it shake overnight for mini preps tomorrow. Luciferase assay on Luc14, GAL4-EED+dox, GAL4-EED 1-3 Wang lab has some old Taq polymerases that expired about 2 years ago. I'm testing them by amplifying g033 cloned into pX330 using the sequencing primers P139 and P140. If any of these polymerases work, I can use them to add dATPs to the ends of my Phusion PCR product for topo cloning. Sample # Polymerase Buffer 1 2x taq mm N/A 2 2x taq mm* N/A 3 2x taq mm** N/A 4 long amp taq LongAmp 5x buffer 5 T4 DNA polymerase Stand taq Mg-free 10x buffer*** 6 Taq DNA polymerase Stand taq Mg-free 10x buffer '*left this out for a long time, this one has more in it '**left this out for a long time, this one has less in it '***This is not the ideal buffer for this polymerase Reactions 1-2 Thing amount Taq MM 2x 5 F primer 0.3 R primer 0.3 template 0.3 H2O 4 Reaction 4 Thing amount LongAmp 5x buffer 2 dNTPs 0.3 F primer 0.3 R primer 0.3 template 0.3 H2O 6.5 long amp taq 0.3 Reaction 5 Thing amount Stand taq Mg-free 10x buffer 1 dNTPs 0.3 F primer 0.3 R primer 0.3 template 0.3 H2O 7.5 T4 DNA polymerase 0.3 Reaction 6 Thing amount Stand taq Mg-free 10x buffer 1 dNTPs 0.3 F primer 0.3 R primer 0.3 template 0.3 H2O 7.5 Taq DNA polymerase 0.3