MIT iGEM Restriction Digests: Difference between revisions
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==Method== | ==Method== | ||
'''The following | '''The following volumes apply to a 20µl analytical digest; for larger, preparative digests, simply scale up (eg. for a 30µl digest, use 3µl of 10x buffer''' | ||
#Add 20µlTotalVolume-(µlDNA + µlBuffer +µlBSA +µlEnzyme)µl ddH2O to PCR tube. | #Add 20µlTotalVolume-(µlDNA + µlBuffer +µlBSA +µlEnzyme)µl ddH2O to PCR tube. (eg. 20µl-(1µlDNA+2µlBuffer+0.2µlBSA+0.5µlEnzymeA+0.5µlEnzymeB)=15.8µl ddH2O) | ||
#Add 0.2µl BSA to tube. | #Add 0.2µl BSA to tube. | ||
#Add 2.0µl 10x Buffer to tube. | #Add 2.0µl 10x Buffer to tube. |
Revision as of 09:51, 24 June 2008
Restriction Digest protocol
Materials
- DNA; the thing you want to cut.
- Appropriate NEB 10x Buffer (check the NEB enzyme chart or catalogue to find compatible buffers).
- Appropriate enzymes.
- ddH2O
- BSA (100x from NEB)
Method
The following volumes apply to a 20µl analytical digest; for larger, preparative digests, simply scale up (eg. for a 30µl digest, use 3µl of 10x buffer
- Add 20µlTotalVolume-(µlDNA + µlBuffer +µlBSA +µlEnzyme)µl ddH2O to PCR tube. (eg. 20µl-(1µlDNA+2µlBuffer+0.2µlBSA+0.5µlEnzymeA+0.5µlEnzymeB)=15.8µl ddH2O)
- Add 0.2µl BSA to tube.
- Add 2.0µl 10x Buffer to tube.
- Add appropriate amount of DNA to tube.
- Add 0.5µl of each enzyme to tube.
- MIX THE REACTION BY PIPETTING HALF THE VOLUME UP AND DOWN.
- Incubate the reaction at 37*C for 2hrs to ensure complete digestion.
- Deactivate the enzymes by heating for 80*C for 20min (in the thermocycler). This will inactivate most enzymes, though check to make sure that your enzyme can be heat deactivated by checking in the NEB catalogue.
- Keep digest at -20*C or run immediately on gel.