Template:Team:Groningen/CONTENT/EXPERIMENTS/tasA PCR (t2)
00:00, 13 May 2015 - 00:00, 13 May 2015
B. subtilis 168 DNA was used.
dNTP Mastermix (MM) was prepared with a final concentration of 2 mM of each dNTP by adding water, see Table 1.
Compound
Amount
100 mM dATP
20 µL
100 mM dTTP
20 µL
100 mM dCTP
20 µL
100 mM dGTP
20 µL
\( \mathrm{H_2O}\)
920 µL
Preparation Mastermix.
The Mastermix with 2 mM of each dNTP is added to buffer, phusion polymerase and water in the amounts indicated in Table 2.
Compound
Amount
5x buffer
33 µL
dNTP MM (2 mM)
16.5 µL
Phusion polymerase
1.65 µL
\( \mathrm{H_2O}\)
112.2 µL
Mastermix for PCR 1.
The resulting solution was combined with template DNA and primers. See Table 3.
Compound
Amount
Mastermix PCR 1
30 µL
Template DNA
0.3 µL
Each primer
0.3 µL
Components per sample.
The following samples were made. See Table 4.
#
Primer 1
Primer 2
Template DNA
3
5 tasA for
10 PstI overlap rev
B. subtilis 168
4
9 PstI overlap for
8 EcoRI overlap rev
B. subtilis 168
5
7 EcoRI overlap
6 tasA rev
B. subtilis 168
Components per sample.
#
Step
Temperature
Time
1
Initial denaturation
98 °C
2:00
2
Denaturation
98 °C
0:10
3
Annealing
60 °C
0:20
4
Extension
72 °C
0:30
5
Go back to step 2 (30x)
6
Final extension
72 °C
5:00
PCR Thermocycle.
The samples were loaded on 1% agarose gel with EtBr to check for PCR products. The results are listed in Table 6.
#
Product length
3
± 100 bp
4
± 150 bp
5
± 700 bp
PCR Results.
As the product at ± 700 bp was not that clearly visible, the PCR was repeated.