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Team:University College London/Protocols/RestrictionEnzymeDigest1
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<html><img href="https://static.igem.org/mediawiki/2012/d/de/Ucl2012-protocols-logo-enzdig.png" /></html> | <html><img href="https://static.igem.org/mediawiki/2012/d/de/Ucl2012-protocols-logo-enzdig.png" /></html> | ||
| - | == Enzyme Digest Protocol 1 == | + | == Enzyme Digest Protocol 1 ==</noinclude> |
| - | </noinclude> | + | |
'''Step 1 - Thawing cells:''' Thaw all materials on ice | '''Step 1 - Thawing cells:''' Thaw all materials on ice | ||
'''Step 2 - Adding Ingredient:''' Add the following ingredients to autoclaved/sterile eppendorf tubes | '''Step 2 - Adding Ingredient:''' Add the following ingredients to autoclaved/sterile eppendorf tubes | ||
| - | + | {| class="wikitable" | |
| + | |- | ||
| + | ! 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 3 - Addition of BioBrick:''' Flick contents gently and centrifuge. | ||
Latest revision as of 01:36, 27 September 2012
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
