Team:Groningen/Notebook/tasA Colony PCR t74
Blue Bio Energy
tasA Colony PCR (t74)
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 after the colony PCR did not show the correct bands, indicating that the ligation of tasA with PliaG did not work.
Colony PCR
00:00, 18 August 2015 - 00:00, 18 August 2015
A new colony PCR was performed with 8 colonies from the T2+PliaG plate and control (T2). Each colony was resuspended in 20 µL MQ water. A Mastermix was made for 9 samples.
Component
Amount
Phusion polymerase
2 µL
5x phusion buffer
36 µL
DNTP MM (2 mM)
18 µL
\( \mathrm{H_2O}\)
120 µL
Primers (pSB1C3 colony PCR)
3 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
98 °C
10:00
2
Denaturation
95 °C
0:30
3
Annealing
55 °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 loaded on a 1% agarose gel with DNA stain G 1:50000. On the gel it was seen that the insert did not have the correct size, meaning that the promoter (PliaG) did not ligate in T2.