Template:Team:Groningen/CONTENT/EXPERIMENTS/tasA PCR (t4)
00:00, 15 May 2015 - 00:00, 15 May 2015
B. subtilis 168 DNA was used.
The Mastermix (MM) with 2 mM of each dNTP is added to buffer, phusion polymerase and water in the amounts indicated in Table 7.
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 2.
The resulting solution was combined with template DNA and primers. See Table 8.
Compound
Amount
Mastermix PCR 1
30 µL
Template DNA
0.3 µL
Each primer
0.3 µL
Components per sample.
#
Primer 1
Primer 2
Template DNA
5
7 tasA EcoRI overlap for
6 tasA rev
B. subtilis 168
6
5 tasA for
8 tasA EcoRI overlap rev
PCR product 1.3 + 1.4
7
9 tasA PstI overlap for
6 tasA rev
PCR product 1.4 + 1.5
8
5 tasA for
8 tasA EcoRI overlap rev
PCR product 1.3 + 1.4 + 1.5
1
5 tasA for
8 tasA EcoRI overlap rev
PCR product 1.3
Components per sample.
Sample 1 is a control to check for genomic DNA of PCR 1.
#
Step
Temperature
Time
1
Initial denaturation
98 °C
3:00
2
Denaturation
98 °C
0:30
3
Annealing
62 °C
0:30
4
Extension
72 °C
1:00
5
Go back to step 2 (3x)
6
Denaturation
98 °C
0:30
7
Annealing
60 °C
0:30
8
Extension
72 °C
1:00
9
Go back to step 6 (3x)
10
Denaturation
98 °C
0:30
11
Annealing
58 °C
0:30
12
Extension
72 °C
1:00
13
Go back to step 10 (3x)
14
Denaturation
98 °C
0:30
15
Annealing
56 °C
0:30
16
Extension
72 °C
1:00
17
Go back to step 14 (3x)
18
Denaturation
98 °C
0:30
19
Annealing
54 °C
0:30
20
Extension
72 °C
1:00
21
Go back to step 18 (3x)
22
Denaturation
98 °C
0:30
23
Annealing
54 °C
0:30
24
Extension
72 °C
1:00
25
Go back to step 22 (30x)
26
Final extension
72 °C
10:00
PCR Thermocycle.
#
Product length
5
± 700 bp
6
± 250 bp
7
no clear product
8
no clear product
1
± 250 bp
PCR Results.
Sample 7 should have showed a band at ± 850 bp and sample 8 at ± 950 bp, but as this was not observed, the product was discarded. In sample 1 still genomic DNA was present. For further PCR experiments, a gelpurification of the PCR product was done to get rid of the genomic DNA. Sample 1 was discarded, as it had no use in further experiments.