Team:Goettingen/protocol PCR

From 2014.igem.org

PfuS PCR

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:

CyclesReactionTemp.Time
1Pre-running98 °C10 min
30Denature9810 s
30Annealing54-60 °C1 min
30Primer Extension72 °C15 s - 2.5 min
1Final elongation72 °C10 min
1Hold4 °C


Watch the following tutorial to learn about the whole process:


HERE you can find the high quality video.



Fusion PCR

Reaction protocol 50 µl:

4 μlPrimer forward (5 pmol)
4 μlPrimer reverse (5 pmol)
100 μltemplate-DNA of each fragment
10 μl5x HF-Buffer
1 μlPfuS-Polymerase
2 μldNTPs (12.5 mM)
add 50 μlsterile water
Amount of cyclesReaction Temp. Time
1Pre-running98°C1 min
10Denaturation98°C15 sec
10Annealing52°C30 sec
10Primer Extension 72°C2 min
1Pause8°Cadd primer
21Denaturation98°C15 sec
21 Annealing52°C30 sec
21Primer Extension72°C2 min + 5 sec/cycle
1Final elongation72°C10 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.