Team:Wash U/Protocol
From 2009.igem.org
(Difference between revisions)
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:6. Transfer the tubes to an incubator set at 80C for another 20 minutes. This step will deactivate the restriction enzymes.<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> | :7. Digestion is now finished and products should be stored at -20C.<br> | ||
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'''Ligation''' | '''Ligation''' | ||
- | : | + | :Note: this procedure requires the products of a successful digestion.<br> |
- | + | :1. Thaw the 10X T4 DNA Ligase Reaction Buffer and mix to dissolve the precipitate.<br> | |
- | + | :2. Add 11 uL of dH2O to a 200 uL PCR tube. Then add 2 uL of each of the digestion products (upstream, downstream and destination) together into this new tube.<br> | |
+ | :3. Add 2 uL of 10X T4 DNA Ligase Reaction Buffer to the 200 uL PCR tube.<br> | ||
+ | :4. Add 1 uL DNA Ligase to the PCR tube and flick to ensure the contents are mized.<br> | ||
+ | :5. Let the mix stand for 10 minutes at room temperature before incubating at 80C for 20 minutes (deactivates enzymes).<br> | ||
+ | :6. Store the products at -20C until they are needed for a transformation.<br> | ||
Revision as of 18:24, 30 June 2009