Template:Team:Groningen/CONTENT/LOGBOOK/PCR pg10
PCR (pg10)
An overlap PCR was done to combine the two separate parts of the PGA gene.
Make a more negatively charged biofilm.
A band of 3000 bp can be seen.
<a class="postscriptum protocol" href="https://2015.igem.org/Team:Groningen/Protocols_and_Protocols/PCR">PCR</a>
12:00, 21 July 2015 - 13:00, 21 July 2015
Ordered PGA from IDT in 2 parts was PCRed to obtain the whole PGA. Used an decreasing annealing temp after each 5 cycli.
The following PCR mix was prepared.
Compound
Amount
Dreamtaq polymerase
1 µL
Dreamtaq 10x buffer
2 µL
Template DNA
1 µL
Each primer
0.3 µL
\( \mathrm{H_2O}\)
16 µL
PCR components.
The following thermocycle was used for the PCR.
#
Step
Temperature
Time
1
Initial denaturation
98 °C
5:00
2
Denaturation
98 °C
0:10
3
Annealing
75 °C
0:30
4
Extension
72 °C
2:00
5
Go back to step 2 (5x)
6
Denaturation
98 °C
0:10
7
Annealing
60 °C
0:30
8
Extension
72 °C
2:00
9
Go back to step 6 (5x)
10
Denaturation
98 °C
0:10
11
Annealing
58 °C
0:30
12
Extension
72 °C
2:00
13
Go back to step 10 (5x)
14
Denaturation
98 °C
0:10
15
Annealing
55 °C
0:30
16
Extension
72 °C
2:00
17
Go back to step 14 (5x)
18
Denaturation
98 °C
0:10
19
Annealing
50 °C
0:30
20
Extension
72 °C
2:00
21
Go back to step 18 (5x)
22
Denaturation
98 °C
0:10
23
Annealing
45 °C
0:30
24
Extension
72 °C
2:00
25
Go back to step 22 (5x)
26
Final extention
72 °C
10:00
PCR Thermocycle.
PCR product was loaded (1 µL product + 4 µL H2O + 1 µL 6X loading buffer) on 1% agarose gel with 1:50000 SERVA DNA Stain G and gel was ran for 30 min at 100 V. Multiple bands visible on the gel.