Haynes Lab:Notebook/CRISPR Editing/2014/09/20: Difference between revisions

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==07//2014==
==09/20/2014==
Ran gel of gradient +/- DMSO PCR reactions from yesterday<br>
Seems obvious that DMSO is necessary for PCR to work. Interesting that the higher annealing temperatures work better than lower ones. Start seeing two bands closer to 56°C.




<br>
PCR of gRNA 20 samples<br>
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Samples'''
|-
| gRNA 20 + dox 1
|-
| gRNA 20 + dox 2
|-
| gRNA 20 - dox 1
|-
| gRNA 20 - dox 2
|-
| gRNA 20 Luc 14
|}
Master mix 1
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''1 rxn'''
| align="center" style="background:#f0f0f0;"|'''5.5 rxns'''
|-
| water||23.5||129.25
|-
| 5x buffer||10||55
|-
| dNTPs||1||5.5
|-
| FP||2.5||13.75
|-
| RP||2.5||13.75
|-
| DMSO||1.5||8.25
|-
| ||41||
|}
add 41ul of MM 1 to each tube<br>
add 4ul of each template to each tube (400ng)
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''1 rxn'''
| align="center" style="background:#f0f0f0;"|'''5.5 rxns'''
|-
| H20||4.5||24.75
|-
| Phusion||0.5||2.75
|}
add 5 ul of MM 2 to each tube<br>
PCR <br>
:98°C for 3 min
:36 cycles
::98°C for 10s
::66°C for 30s
::72°C for 60s
:72°C for 10 min
:4°C forever


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Revision as of 15:11, 21 September 2014

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09/20/2014

Ran gel of gradient +/- DMSO PCR reactions from yesterday
Seems obvious that DMSO is necessary for PCR to work. Interesting that the higher annealing temperatures work better than lower ones. Start seeing two bands closer to 56°C.



PCR of gRNA 20 samples

Samples
gRNA 20 + dox 1
gRNA 20 + dox 2
gRNA 20 - dox 1
gRNA 20 - dox 2
gRNA 20 Luc 14

Master mix 1

' 1 rxn 5.5 rxns
water 23.5 129.25
5x buffer 10 55
dNTPs 1 5.5
FP 2.5 13.75
RP 2.5 13.75
DMSO 1.5 8.25
41

add 41ul of MM 1 to each tube
add 4ul of each template to each tube (400ng)

' 1 rxn 5.5 rxns
H20 4.5 24.75
Phusion 0.5 2.75

add 5 ul of MM 2 to each tube

PCR

98°C for 3 min
36 cycles
98°C for 10s
66°C for 30s
72°C for 60s
72°C for 10 min
4°C forever


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