Team:Groningen/Notebook/tasA PCR t28
Blue Bio Energy
tasA PCR (t28)
In this experiment tasA was PCR'ed to increase the amount of DNA.
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.
On gel, the band for tasA was hardly visible, but the PCR possibly did work.
PCR
00:00, 8 June 2015 - 00:00, 8 June 2015
In this experiment it was attempted to increase the amount of tasA. For this first a Mastermix was made, to which primer and DNA were added.
Component
Amount
10x buffer
10 µL
dNTP MM (2 mM)
10 µL
Dreamtaq polymerase
1 µL
\( \mathrm{H_2O}\)
76,5 µL
Mastermix PCR.
Component
Amount
Mastermix PCR 5
30 µL
tasA DNA (4.2)
0.3 µL
5 tasA for
0.3 µL
6 tasA rev
0.3 µL
Components PCR sample.
The following PCR cycle was used.
#
Step
Temperature
Time
1
Initial denaturation
98 °C
3:00
2
Denaturation
98 °C
0:30
3
Annealing
60 °C
0:30
4
Extension
72 °C
1:00
5
Go back to step 2 (repeat 30x)
6
Final extension
72 °C
10:00
PCR thermocycle.
The PCR prduct was loaded on a 1% agarose gel with DNA stain G (1:50000) and run for 1 hour on 100 V with the following samples.
Sample:
30 µL DNA (tasA).
10 µL 6x buffer.
Ladder:
10 µL NEB ladder.
The band of tasA was hardly visible on gel.