Digest Standard Protocol

Digests are used to cut your DNA with restriction enzymes.

We always used buffers and enzymes from New England Biolabs, whose website can be found [http://www.neb.com/nebecomm/default.asp HERE]. Their site has great tools that can be used to find compatible enzymes, determine which buffers to use and whether to use BSA or not.

Protocol

1. Generally, use 20 μL for total digest volume

• If linearizing a plasmid, use more than 20 μL

2. NEVER use more than 10% enzyme

• Even with a 40 μL digest, you can use 1 μL of enzyme for an overnight digest

3. Add the correct buffer for your enzyme

• Buffers are 10X, so use 1 μL for each 10 μL total volume

4. Check to see if your digest requires BSA

• BSA is 10X, so use 1 μL for each 10 μL total volume

5. Balance of digest mixture should be your vector (DNA you wish to cut)

6. ALWAYS digest in incubator

• Usually leave overnight, but at least 4 hours

Following these guidelines, your digest mixtures should look similar to these:

EXAMPLE DIGEST 1: one enzyme w/ BSA
15 μL Vector
2 μL NEBuffer 3
2 μL BSA
1 μL EcoRV

EXAMPLE DIGEST 2: one enzyme w/ no BSA
17 μL Vector
2 μL NEBuffer 1
1 μL NAE1