Team:UIUC-Illinois/Notebook/Protocols/Digestions
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- | <center><h1> | + | <center><h1>Digestions</h1></center><br/> |
+ | Reaction Mix:<br/><br/> | ||
+ | - 500 ng – Template<br/> | ||
+ | - 5 µl – 10x Buffer<br/> | ||
+ | - 1 µl – NEB Enzyme 1 (10 Units/µl) = 10 Units in 50 µl reaction<br/> | ||
+ | - 1 µl – NEB Enzyme 2 (10 Units/µl) = 10 Units in 50 µl reaction<br/> | ||
+ | - dH2O to 50 µl<br/> | ||
+ | - TOTAL – 50 µl<br/><br/> | ||
+ | Procedure:<br/><br/> | ||
+ | 1. Calculate how much template is needed for digestion. Then calculate how much water needs to be added to make a 50 µl reaction.<br/> | ||
+ | 2. Pipette appropriate amounts of dH20, template, and 10x Buffer in that order into a PCR tube (200 µl). Mix reagents together by vortexing and then tapping tube on desk to keep reagents on the bottom of the PCR tube.<br/> | ||
+ | 3. Add appropriate amounts of Enzyme 1 and Enzyme 2. Once added gently swirl around reaction mix with pipette tip.<br/> | ||
+ | 4. Incubate at 37o C for 1 – 2 hours<br/> | ||
+ | 5. Incubate at 80 oC for 20 minutes to deactivate the restriction enzymes <br/> | ||
+ | 6. Run a gel of your digestions on a gel with low temperature melting agarose. Run at 100V for 1 hr. 30 min.<br/> | ||
+ | 7. Look at gel under 320nm UV light. Conduct gel extraction purification.<br/><br/> | ||
+ | NOTE: When digesting your vector it may be useful to use 2 µl of each enzyme in the 50 µl reaction and extending incubation time.<br/><br/> | ||
</div> | </div> | ||
Latest revision as of 15:19, 4 June 2012