Team:Goettingen/protocol PCR

Fusion PCR

Reaction protocol 50 µl:
      4 μl Primer forward (5 pmol)
      4 μl Primer reverse (5 pmol)
      100 ng template-DNA of each fragment
      10 μl 5x HF-Buffer
      1 μl PfuS-Polymerase
      2 μl dNTPs (12.5 mM)
      ad. 50 μl sterile water

PCR cycles:

Amount of cycles	Reaction	Temp.	Time
1	Pre-running	98°C	1 min
10	Denaturation	98°C	15 sec
10	Annealing	52°C	30 sec
10	Primer Extension	72°C	2 min
1	Pause	8°C	add primer
21	Denaturation	98°C	15 sec
21	Annealing	52°C	30 sec
21	Primer Extension	72°C	2 min + 5 sec/cycle
1	Final elongation	72°C	10 min
1	Hold	4°C	∞

Pfus PCR

Watch the following tutorial to learn about the whole process:

Reaction protocol for 50 µl
      1 μl Primer fwd (5 pmol)
      1 μl Primer rev (5 pmol)
      1 μl Matrizen-DNA (ca. 100 ng)
      10 μl 5x HF-Polymerase Puffer
      0.5 μl PfuS-Polymerase (50 ng)
      1 μl dNTPs (12.5 μmol ml-1)
      35.5 μl dest. Water

PCR cycles:

Cycles	Reaction	Temp.	Time
1	Pre-running	98 °C	10 min
30	Denature	98	10 s
30	Annealing	54-60 °C	1 min
30	Primer Extension	72 °C	15 s - 2.5 min
1	Final elongation	72 °C	10 min
1	Hold	4 °C	∞

Assembly PCR

Reaction protocol 100 µl:
      20 μl 5 x PCR buffer
      4 μl dNTP-Mix(12.5 mM each)
      8 μl Forward oligo
      8 μl Reverse oligo
      100 ng Region I
      100 ng Region II
      100 ng Region III
      ad. 100 μl sterile water

PCR cycles:

Amount of cycles	Reaction	Temp.	Time
1	Initial Denaturation	98°C	10 min
10	Denaturation	98°C	10 sec
10	Annealing	52°C	30 sec
10	Elongation	72°C	2 min
1	Hold	12°C	add oligos
21	Denaturation	98°C	10 sec
21	Annealing	47°C	1 min
21	Elongation	72°C	2 min
1	Final elongation	72°C	10 min
1	Hold	12°C	∞