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

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(Autocreate 2014/09/20 Entry for Haynes_Lab:Notebook/CRISPR_Editing)
 
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==07//2014==
==09/20/2014==


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 15:08, 21 September 2014

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09/20/2014

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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