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Team:Columbia-Cooper-NYC/Digestion
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Latest revision as of 03:42, 4 October 2012
Digestion Protocol for 3A Assembly Method
- The following was protocol provided by bioworks:
- Determine upstream part, downstream part and destination plasmid
- Label three PCR tubes as U, D, P respectively
- To each tube add 500ng of plasmid to be digested
- Add necessary amount of water so final volume is 42.5µl
- Add 5µl of NEBuffer 2 to each tube
- Add 0.5µl of BSA to each tube
- Add 1µl of first appropriate enzyme
- Add 1µl of second appropriate enzyme
- Ensure that digest is well mixed
- Incubate the digests at 37C for 15 minutes in thermocycler
- Incubate the digests at 80C for 20 minutes to deactivate the restriction enzymes
- Store digest at -20C
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