Template:Team:Groningen/CONTENT/EXPERIMENTS/tasA Assembly PCR (t6)
00:00, 18 May 2015 - 00:00, 18 May 2015
The following PCR was performed.
Compound
Amount
5x buffer
28 µL
dNTP MM (2 mM)
14 µL
Phusion polymerase
1.4 µL
\( \mathrm{H_2O}\)
95.2 µL
Mastermix for PCR 3.
Compound
Amount
Mastermix PCR 3
30 µL
Template DNA
0.3 µL
Each primer
0.3 µL
Components per sample.
#
Primer 1
Primer 2
Template DNA
3
5 tasA for
6 tasA rev
PCR product 2.5 + 2.6
4
5 tasA for
6 tasA rev
PCR product 1.3 + 1.4 + 2.5
Components per sample.
#
Step
Temperature
Time
1
Initial denaturation
98 °C
3:00
2
Denaturation
98 °C
0:30
3
Annealing
56 °C
0:30
4
Extension
72 °C
1:00
5
Go back to step 2 (repeat 15x)
6
Denaturation
98 °C
0:30
7
Annealing
70 °C
0:30
8
Extension
72 °C
1:00
9
Go back to step 6 (repeat 15x)
10
Final extension
72 °C
10:00
PCR Thermocycle.
The PCR product was stored in the fridge at 4 °C.
00:00, 19 May 2015 - 00:00, 19 May 2015
A gel was run with the PCR products on 1% agarose gel with EtBr.
On the gel, for sample 3 two bands were visible, indicating that fragment 2.5 and 2.6 were not assembled. Sample 4 had no product, so also the PCR assembly of product 1.3, 1.4 and 1.5 did not succeed.