USTC/Notebook/Restriction Digests
From 2008.igem.org
Restriction Digests
Materials
- DNA; the thing you want to cut. Usually plasmid or PCR product. Measure concentration in Nanodrop beforehand.
- Appropriate NEB 10x Buffer (check the NEB enzyme chart or catalogue to find compatible buffers).
- Appropriate enzymes.
- ddH2O
- BSA (100x from NEB)
pr
The following volumes apply to a 20µl analytical digest; for larger, preparative digests, simply scale up (eg. for a 30µl digest, use 3µl of 10x buffer, etc)
add the following components to PCR tube:
- 2µl BSA
- 2µl 10x Buffer
- appropriate amount of DNA
- 0.5µl of each enzyme
- appropriate amount of ddH2O to PCR tube. (20µlTotalVolume-(µlDNA + µlBuffer +µlBSA +µlEnzyme)µleg. 20µl-(1µlDNA+2µlBuffer+0.2µlBSA+0.5µlEnzymeA+0.5µlEnzymeB)=15.8µl ddH2O)
- mix the reaction by stiring
- Incubate the reaction at 37*C for 2hrs to ensure complete digestion.
- Store digest at -20*C or run immediately on gel.