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Team:University College London/Protocols/RestrictionEnzymeDigest1
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Revision as of 01:36, 27 September 2012 by Rwilkinson (Talk | contribs)
Enzyme Digest Protocol 1
Step 1 - Thawing cells: Thaw all materials on ice
Step 2 - Adding Ingredient: Add the following ingredients to autoclaved/sterile eppendorf tubes
| Component | Amount (ul) (one enzyme used) | Amount (ul) (two enzymes used) |
|---|---|---|
| dH20 | 2.5 | 1.5 |
| Buffer 1x | 1 | 1 |
| DNA template | 5 | 5 |
| BSA | 0.5 | 0.5 |
| Enzyme 1 | 1 | 2 |
| Enzyme 2 | N/A | 1 |
Step 3 - Addition of BioBrick: Flick contents gently and centrifuge.
Step 4 - Centrifuge:
RPM: 14000
Time: 1 minute
Temperature: 18oC
Step 5 - Digest Program: Place the samples on a thermocycler under the following conditions:
RPM: 550
Time: 2.5 hours
Temperature: 37oC
Step 6 - Denaturing Enzymes: If you are not running the samples on a gel immediately, denature the restriction enzymes by running the samples on a thermocycler under the following conditions:
RPM: 550
Time: 25 minutes
Temperature: 65oC
