Team:Wash U/Protocol
From 2009.igem.org
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:To view the full BioBrick Manual procedures, please click [http://partsregistry.org/Help:Transformation_Protocol here]. | :To view the full BioBrick Manual procedures, please click [http://partsregistry.org/Help:Transformation_Protocol here]. | ||
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+ | '''Mini-Prep''' | ||
+ | :text | ||
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'''Digestion''' | '''Digestion''' | ||
- | : | + | :Note: The procedures from this point forward assume that DNA has been amplified and purified. If this is not the case, please read the Polymerase Chain Reaction procedure for amplification and the Mini-Prep procedure for purification.<br> |
+ | :1. Begin by thawing the upstream, downstream, and destination plasmid parts along with the NEBuffer 2 and BSA.<br> | ||
+ | :2. In three separate PCR microcentrifuge tubes labeled upstream, downstream, and destination, add 500ng of the respective dried DNA and dilute with dH20 to 42.5 uL.<br> | ||
+ | :3. Add 5 uL of NEBuffer 2 and 0.5 uL of BSA to eah tube.<br> | ||
+ | :4. Add 1 uL of the first appropriate enzyme to each tube. Then add 1 uL of the second appropriate enzyme.<br> | ||
+ | :5. Flick each tube to mix reagents and incubate at 37C for 15 minutes.<br> | ||
+ | :6. Transfer the tubes to an incubator set at 80C for another 20 minutes. This step will deactivate the restriction enzymes.<br> | ||
+ | :7. Digestion is now finished and products should be stored at -20C.<br> | ||
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Revision as of 18:08, 30 June 2009