Jonesma Lab:Protocols:colony: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(New page: <h3><font style="color:#F8B603;">Colony PCR</font></h3> ---- #Take a colony and resuspend in 5 μL dH<sub>2</sub>O #Streak the tip on a fresh antibiotic plate and incubate at 37°C to rege...) |
No edit summary |
||
| Line 4: | Line 4: | ||
#Streak the tip on a fresh antibiotic plate and incubate at 37°C to regenerate colony | #Streak the tip on a fresh antibiotic plate and incubate at 37°C to regenerate colony | ||
#Add the following reagents to a tube | #Add the following reagents to a tube | ||
#Setup additional tubes as needed- always include a positive and negative control | |||
#Add tubes to PCR machine, and run with following programme | |||
#Run PCR product out on a gel | |||
<h4>Reagents for PCR</h4> | |||
2 μL 10x PCR buffer | 2 μL 10x PCR buffer | ||
| Line 18: | Line 23: | ||
1 μL Taq | 1 μL Taq | ||
<h4>PCR programme</h4> | |||
# | #94°C 5 min | ||
#94°C 30s | |||
#55°C 30s | |||
#72°C 1min | |||
#Loop to #2 for 35 cycles | |||
#72°C 2min | |||
#10°C indefinitely | |||
Revision as of 14:09, 24 November 2015
Colony PCR
- Take a colony and resuspend in 5 μL dH2O
- Streak the tip on a fresh antibiotic plate and incubate at 37°C to regenerate colony
- Add the following reagents to a tube
- Setup additional tubes as needed- always include a positive and negative control
- Add tubes to PCR machine, and run with following programme
- Run PCR product out on a gel
Reagents for PCR
2 μL 10x PCR buffer
2 μL 1/4 dNTPS
2 μL 20mM MgCl2
6 μL dH2O
1 μL 10μM Forward primer
1 μL 10μM Reverse primer
1 μL Taq
PCR programme
- 94°C 5 min
- 94°C 30s
- 55°C 30s
- 72°C 1min
- Loop to #2 for 35 cycles
- 72°C 2min
- 10°C indefinitely
