Team:Goettingen/protocol PCR

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	∞

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	∞

E.coli colony PCR

1. Pick a fresh colony of about 1 mm and resuspend it in 10 µl water.
2. Use 1 µl as a template for a 25 µl PCR reaction.

Yeast colony PCR

1. Aliquot 20 µl NaOH (20 mM) into 1.5 ml tubes
2. Pick colonies (use pipet tips) into the NaOH
3. Incubate at 95°C for ~ 45 minutes
4. Centrifuge at max speed for 10 minutes
5. Use 5 µl of supernatant as template in a (50 µl) PCR.