Team:Goettingen/protocol DNA

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!)

SEAMLESS Cloning

1. Construct primers for the desired construct with 15 bp overhangs for homologous recombination later.
2. Amplify DNA fragments, purify them and measure concentration.
3. Calculate the amount of PCR product amount you need with the formula:
     ng of insert = (2 x bp of insert x 100 (ng vector)) / bp of vector
4. Pipette 100 ng of vector and the calculated amount of insert together. Scale down the reaction from 20 µl to the smallest volume possible.
5. Add 5 fold buffer and last the 10 fold enzyme mix.
6. Incubate for 30 min at room temperature.
7. Cool the sample on ice for not more than 5 minutes.
8. Use the whole sample for transformation.