Team:Goettingen/protocol PCR
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Revision as of 11:51, 17 October 2014
Overview
PCR Methods
Plasmid Construction
- Restriction of DNA
- Ligation of DNA fragments
- BP recombination reaction
- LR recombination reaction
- SEAMLESS Cloning
- Peptide Library construction
Plasmid Transformation
- E.coli competent cells
- Plasmid isolation (E.coli)
- E.coil transformation
- Plasmid isolation (Yeast)
- Yeast transformation
Colony Scanning
Protein Assessment
In vivo tests
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:
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 | ∞ |
Watch the following tutorial to learn about the whole process:
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.