Difference between revisions of "Team:Freiburg/Protocols/Fusion PCR"

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Latest revision as of 07:09, 20 November 2015

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Fusion PCR

PCR mix I

ingredient
volume
Template
200 - 300 ng
Q5 Polymerase
0.5 µl
dNTPs
4 µl
DMSO
1 µl
Q5 reaction buffer (5x)
10 µl
dH2O
up to 45 µl

PCR run I

temperature
time
98 °C
5 min
98 °C
30 s
** °C
30 s
72 °C
35 s per 1 kb amplicon length

Repeat steps 2 till 4 for 5 times.

** Calculated annealing temperature of the whole owerlap (OligoCalc, basic). If the temperature of step 3 is close to or higher than 72 °C, a touchdown PCR should be performed: The temperature of step 3 will be reduced for 0.5 °C every cycle, starting at 72 °C.

PCR run II

Add 2.5 μl of each primer that anneal at the ends of the new construct.

temperature
time
98 °C
5 min
98 °C
30 s
63 °C
30 s
72 °C
35 s per 1 kb amplicon length
72 °C
10 min

Repeat steps 2 till 4 for 18 times.