Restrictions

Restricting DNA
We have conducted restrictions in varying concentrations and total volumes; however they all follow the same basic procedure. Below are two of the different restrictions we carried out.

Dilute


 * 46μl MillQ H2O


 * 10μl 10 x buffer


 * 40μl plasmid sample


 * 2μl enzyme 1


 * 2μl enzyme 2

Total volume = 100μl

Concentrated


 * 10μl MilliQ H2O


 * 3μl 10 X buffer


 * 10μl plasmid sample


 * 1μl enzyme 1


 * 1μl enzyme 2

Total volume = 30μl

Incubate solutions for 90 minutes in a 37°C water bath.

If DNA is to be run on a gel and a band cut out, this can be done without purifying the DNA. For all other downstream applications or for storage, solution must be purified to remove the enzymes.

Back to Team:Newcastle University/Notebook