Team:Groningen/Notebook/Overlap PCR pg21
Blue Bio Energy
Overlap PCR (pg21)
PGA was ordered in two separate pieces. These two have to be combined. This was attempted with an overlap PCR where two overlapping fragments can be combined.
Combine the two ordered parts of PGA.
Band have the correct size.
PCR
00:00, 28 July 2015 - 00:00, 28 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.