Team:Michigan/Notebook/DigestProtocol

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HOME THE TEAM THE PROJECT REGISTRY PARTS NOTEBOOK


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


EXAMPLE DIGEST 3: two enzymes w/ BSA
14 μL Vector
2 μL NEBuffer BamHI
2 μL BSA
1 μL BamHI
1 μL NotI