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Team:Goettingen/protocol DNA
From 2014.igem.org
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
Restriction of DNA
1. Use up to 1 µg of DNA in a 20 µl reaction volume. Add 1 µl enzyme, 2 µl 10x recommended buffer and add up to 20 µl water.
2. Incubate for at least 1 hour at 37°C (or enzyme specific temperature).
3. Following the incubation, add 1.5 µl SAP (shrimp alkaline phosphatase) to plasmid backbones for 5’‑dephosphorylation and incubate again at 37°C for 10-30 minutes.
4. Stop reaction by heat-inactivation at 65°C for 5 minutes.
Ligation of DNA fragments
1. Measure the concentration of fragments which should be used for the ligation reaction.
2. Calculate the ratio between insert and backbone (1:1) using the following formula:
3. Pipette all things together. Use 2 µl of 10x T4 Ligation buffer (stored at -20°C, ATP containing) and at least 1 µl T4 ligase.
4. Incubate your reaction mix for 1 hour at room temperature or over night at 16°C.
5. Use at least 4 µl of the reaction mixture for transformation. (Previous purification of the ligation product can avoid false positive colonies!)
