Digest Standard Protocol

Digests are used to cut your DNA with restriction enzymes.

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