Template:Team:Groningen/CONTENT/Protocols and Protocols/ColonyPCR

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.