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

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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