Team:Michigan/Notebook/DigestProtocol

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 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 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.

=Digest Protocol=

1. Generally, use 20 &mu;L for total digest volume
 * If linearizing a plasmid, use more than 20 &mu;L

2. NEVER use more than 10% enzyme
 * Even with a 40 &mu;L digest, you can use 1 &mu;L of enzyme for an overnight digest

3. Add the correct buffer for your enzyme
 * Buffers are 10X, so use 1 &mu;L for each 10 &mu;L total volume

4. Check to see if your digest requires BSA
 * BSA is 10X, so use 1 &mu;L for each 10 &mu;L 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 &mu;L Vector 2 &mu;L NEBuffer 3 2 &mu;L BSA 1 &mu;L EcoRV

EXAMPLE DIGEST 2: one enzyme w/ no BSA 17 &mu;L Vector 2 &mu;L NEBuffer 1 1 &mu;L NAE1

EXAMPLE DIGEST 3: two enzymes w/ BSA 14 &mu;L Vector 2 &mu;L NEBuffer BamHI 2 &mu;L BSA 1 &mu;L BamHI 1 &mu;L NotI

=Digest Purification Protocol=

All of our digest purifications are performed using QIAGEN kits and protocols.

QIAGEN's website can be found HERE.