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USTC/Notebook/Restriction Digests
From 2008.igem.org
(Difference between revisions)
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*'''BSA''' (100x from NEB) | *'''BSA''' (100x from NEB) | ||
| - | + | Procedure | |
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, etc) | 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, etc) | ||
add the following components to PCR tube: | add the following components to PCR tube: | ||
| - | *2µl BSA | + | *2µl BSA (diluted to 10X beforehand) |
*2µl 10x Buffer | *2µl 10x Buffer | ||
*appropriate amount of DNA | *appropriate amount of DNA | ||
*0.5µl of each enzyme | *0.5µl of each enzyme | ||
| - | *appropriate amount of | + | *appropriate amount of ddH<sub>2</sub>O to PCR tube. (20µlTotalVolume-(BSA + Buffer + DNA + Enzyme)µl |
| - | + | eg. 20µl-(5µlDNA+2µlBuffer+2µlBSA+0.5µlEnzymeA+0.5µlEnzymeB)=10µl ddH<sub>2</sub>O) | |
*mix the reaction by stiring | *mix the reaction by stiring | ||
*Incubate the reaction at 37*C for 2hrs to ensure complete digestion. | *Incubate the reaction at 37*C for 2hrs to ensure complete digestion. | ||
*Store digest at -20*C or run immediately on gel. | *Store digest at -20*C or run immediately on gel. | ||
Revision as of 16:54, 27 October 2008
Restriction Digests
Materials
- DNA; the thing you want to cut. Usually plasmid or PCR product. Measure concentration in Nanodrop beforehand.
- Appropriate NEB 10x Buffer (check the NEB enzyme chart or catalogue to find compatible buffers).
- Appropriate enzymes.
- ddH2O
- BSA (100x from NEB)
Procedure
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, etc)
add the following components to PCR tube:
- 2µl BSA (diluted to 10X beforehand)
- 2µl 10x Buffer
- appropriate amount of DNA
- 0.5µl of each enzyme
- appropriate amount of ddH2O to PCR tube. (20µlTotalVolume-(BSA + Buffer + DNA + Enzyme)µl
eg. 20µl-(5µlDNA+2µlBuffer+2µlBSA+0.5µlEnzymeA+0.5µlEnzymeB)=10µl ddH2O)
- mix the reaction by stiring
- Incubate the reaction at 37*C for 2hrs to ensure complete digestion.
- Store digest at -20*C or run immediately on gel.
