Restricting Plasmids (Double Restriction)

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.

Total volume = 100μl
 * 46μl MillQ H2O
 * 10μl 10 x buffer
 * 40μl plasmid sample
 * 2μl enzyme 1
 * 2μl enzyme 2

Concentrated Total volume = 30μl
 * 10μl MilliQ H2O
 * 3μl 10 X buffer
 * 10μl plasmid sample
 * 1μl enzyme 1
 * 1μl enzyme 2



The DNA purification kit we use to purify enzymatic reaction mixtures.


 * 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.