Template:Team:Groningen/CONTENT/EXPERIMENTS/tasA Colony PCR (t73)
00:00, 17 August 2015 - 00:00, 17 August 2015
A colony PCR with 6 colonies of the T2+PliaG plate was done. Each colony was taken from the plate and resuspended in 20 µL sterile MQ water. A Mastermix for 16 samples was made.
Component
Amount
Phusion polymerase
4.5 µL
5x phusion buffer
80 µL
DNTP MM (2 mM)
35 µL
\( \mathrm{H_2O}\)
240 µL
VR + VF2 primers
1 µL each
Components Mastermix.
For the PCR samples, 18 µL of Mastermix was added to 4 µL of colony suspension. The following PCR thermocycle was used.
#
Step
Temperature
Time
1
Initial denaturation
99 °C
10:00
2
Denaturation
95 °C
0:30
3
Annealing
58 °C
0:30
4
Extension
72 °C
1:30
5
Go back to step 2 (repeat 30x)
6
Final extension
72 °C
10:00
PCR thermocycle.
The samples were run on a 1% agarose gel with DNA stain G 1:50000. 5 µL of sample DNA was mixed with 1 µL 6x buffer. As a ladder 1 µL of the GeneRuler™ 1 kb DNA Ladder was used. The gel was run for ∼30 minutes at 100 V. No correct PCR product was visible on the gel. All bands are at the same height as the control (T2 without promoter).