Team:Groningen/Protocols and Protocols/ColonyPCR
Blue Bio Energy
ColonyPCR
The protocol for Colony PCR. Colony PCR helps to determine whether the insert DNA in plasmid constructs is present or not in the colonies of interest.
ML-I
Dilution
Dilute each colony in 20 μl H2O.
Making PCR colony mixture
Make a PCR colony mixture for each tested colony according to this table:
Component
20 μl reaction
Final concentration
H2O
Add to 20 μl
10 X Dream taq buffer
2 μl
1x
2 mM dNTPs
2 μl
200 μM each
Forward primer
? μl
0.5 μM
Reverse primer
? μl
0.5 μM
Diluted colonies
2 μl
Dream taq DNA polymerase
0.1 μl
0,5 U/ 20 μL
PCR colony mixture
Mixing PCR colony mix
Gently vortex the samples and spin down.
Thermal cycling of PCR mixture
Place the reactions in a thermal cycler. Perform PCR using recommended thermal cycling conditions:
Cycle step
Temperature
Time
Number of cycles
Initial denaturation
98°C
30 seconds
1
Denaturation
98°C
5-10 seconds
25
Annealing
55°C
20 seconds
25
Extension
72°C
15-30 seconds/kb
25
Final extension
72°C
5-10 minutes
1
Hold
20°C
hold
1
Thermal cycle
Checking colony PCR
Check colony PCR samples by performing a gel electrophoresis according to its protocol.