Haynes Lab:Notebook/CRISPR Editing/2015/01/27: Difference between revisions

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<br><br>
Luciferase assay on Luc14, GAL4-EED+dox, GAL4-EED 1-3
<br><br>
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.<br>
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Sample #'''
| align="center" style="background:#f0f0f0;"|'''Polymerase'''
| align="center" style="background:#f0f0f0;"|'''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<br>
'**left this out for a long time, this one has less in it<br>
'***This is not the ideal buffer for this polymerase<br>
Reactions 1-2
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Thing'''
| align="center" style="background:#f0f0f0;"|'''amount'''
|-
| Taq MM 2x||5
|-
| F primer||0.3
|-
| R primer||0.3
|-
| template||0.3
|-
| H2O||4
|}
Reaction 4
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Thing'''
| align="center" style="background:#f0f0f0;"|'''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
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Thing'''
| align="center" style="background:#f0f0f0;"|'''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
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Thing'''
| align="center" style="background:#f0f0f0;"|'''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
|}
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Revision as of 17:14, 27 January 2015

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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



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