PROTOCOL 007: RESTRICTION ENZYME DIGESTION
(10/2012)
REAGENTS
DNA
Water
10X Buffer stock
100X BSA stock
(optional)
Enzyme
PROCEDURE
With rare exception,
our lab uses enzymes from New England Biolabs.
After deciding which
enzyme you will use, determine the following:
á
What is the
appropriate buffer: NEB 1, 2, 3, 4 or U (indicates enzyme-specific buffer)?
á
Is BSA
required?
á
Are other
co-factors required?
á
What is the
appropriate incubation temperature?
á
Optional: Can
the enzyme be heat inactivated?
Remove enzyme and
appropriate buffer from -20¡ C and immediately place enzyme on
ice.
Thaw buffer and BSA,
and mix well by vortexing.
Spin down buffer and
BSA.
It may be necessary
to spin down enzyme but never vortex
it. If spinning is necessary, it should be performed quickly and the enzyme
should be placed back on ice.
Determine 1) the
amount of DNA to be digested and 2) the final volume of digest.
Add components in the
following order to an Eppendorf tube (Note: the example assumes 5 microL of a 0.2 microgram/ microL
DNA stock in a final volume of 30 microL):
1)
Water 20.7
2)
10X buffer 3
3)
DNA 5
4)
100X BSA 0.3
5)
Enzyme 1
Briefly spin
Eppendorf tube and using a P20 pipette set to 10 microL,
gently mix reaction components.
Incubate at the
appropriate temperature for at least one hour and as long as overnight.
Proceed with
downstream application (gel analysis, cloning, etc.)
NOTE: IF A DOUBLE DIGEST IS REQUIRED, REFER TO NEBÕS DOUBLE
DIGEST FINDER FOR THE APPROPRIATE BUFFER AND CONDITIONS:
https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder