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:

 

       What is the appropriate buffer: NEB 1, 2, 3, 4 or U (indicates enzyme-specific buffer)?

       Is BSA required?

       Are other co-factors required?

       What is the appropriate incubation temperature?

       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 NEBS DOUBLE DIGEST FINDER FOR THE APPROPRIATE BUFFER AND CONDITIONS:

 

https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder