Team:Groningen/Notebook/tasA PCR t9
Blue Bio Energy
tasA PCR (t9)
PCR was performed to amplify tasA.
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.
Sample 1 was not assembled correctly; sample 2 did show a band on gel with the right size.
PCR
00:00, 19 May 2015 - 00:00, 19 May 2015
The following PCR was performed.
Compound
Amount
5x buffer
14 µL
dNTP MM (2 mM)
7 µL
Phusion polymerase
0.7 µL
\( \mathrm{H_2O}\)
47.6 µL
Mastermix for PCR 4.
Compound
Amount
Mastermix PCR 4
30 µL
Template DNA
0.3 µL
Each primer
0.3 µL
Components per sample.
#
Primer 1
Primer 2
Template DNA
1
5 tasA for
6 tasA rev
tasA DNA 1
2
5 tasA for
6 tasA rev
tasA DNA 2
Components per sample.
#
Step
Temperature
Time
1
Initial denaturation
98 °C
3:00
2
Denaturation
98 °C
0:30
3
Annealing
55 °C
0:30
4
Extension
72 °C
0:30
5
Go back to step 2 (30x)
6
Final extension
72 °C
10:00
PCR Thermocycle.
The product was stored in the freezer at -20 °C.
00:00, 20 May 2015 - 00:00, 20 May 2015
The PCR products were stored as tasA DNA 1 & tasA DNA 2 and PCR 4 prod 20-5-2015 (side). On gel it was checked if the PCR had worked out. A gel was made of 1% agarose with EtBr. The loaded samples consisted of the components listed below. The samples were compared with 3 µL of DNA ladder.
Sample:
1.5 µL 6x loading buffer
2.5 µL \( \mathrm{H_2O}\)
2 µL sample
Ladder:
3 µL GeneRuler™ 1 kb DNA Ladder
On the gel there were no bands visible for sample 1. Apparently the tasA product had not formed. The gel of sample 2 did show a product at ± 950 bp. This fragment was checked with restriction enzymes to find out if the restriction sites were absent in the tasA gene.