Team:Groningen/Notebook/tasA Colony PCR t73
Blue Bio Energy
tasA Colony PCR (t73)
The colony PCR is performed to check the insert of the E. coli.
Our goal is to overexpress TasA by adding a second copy of the tasA gene under the control of a salt inducible promoter. This overexpression will lead to a stronger biofilm when the biofilm is exposed to salt water.
The gel of the colony PCR showed no right inserts.
Colony PCR
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).