Basic rules:
Amount of enzyme should
never exceed 1/10 of reaction volume, might result in star activity
1 Unit of enzyme cuts 1µg of
DNA in 1h at 37¡C
use 1.5 - 2x more enzyme
than necessary, e.g. for 5µg of DNA use 7.5-10U of enzyme
most enzymes have conc
of 10U/µl, but several are also
20U/µl, check before using them, adding too much enzyme might result in star
activity as well even if volume is lower than 1/10
use reaction volume between
10-100µl
some enzymes need BSA in
reaction, prepare 10x BSA from 100x BSA stock, use like NEB Restriction enzyme
buffers
Restriction digest for screening Mini-prep DNA:
Use 1-2µl of Mini-Prep DNA
and 0.1-0.2µl of Restriction enzyme
Example: 6 clones
Mastermix:
6.5x
1.5µl
plasmid
1µl
10xNEBuffer 2 6.5µl
1µl
10x BSA 6.5µl
0.2µl
NheI 10U/µl 1.3µl
0.1µl
XhoI 20U/µl 0.65µl
6.2µl
H2O 40.3µl
add 8.5µl Mastermix to 1.5µl
plasmid for 10µl final volume, incubate 1h at 37¡C,
load everything on agarose gel
Anne Schohl, 2008