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Team:TU Darmstadt/Protocols/Restriction digest
From 2012.igem.org
Contents |
Restriction digest
About
In order to insert DNA fragments into plasmids via ligation it is necessary to digest both components with restriction enzymes
Procedure
DNA Digestion (50 µL batch)
Add up the following:
- 10x NEBuffer 5 µL
- DNA 1 µg
- Restriction Enzyme (10 u) 1 µL
- 100x BSA 0.2 µL
- Add dH2O for total reaction volume of 50 µL
- Incubate for 1 h at an enzyme specific temperature
- inactivate enzymes by incubating at 65°C for 20 min
Single Restriction Enzyme Digests
An analytical restriction enzyme reaction is usually performed in a volume of approximately 20µl on 0.2-1.5µg of substrate DNA using a 2- to 10-fold excess of enzyme over DNA, based on unit definition. Use of an unusually large volume of DNA or enzyme may give aberrant results. Caution should be exercised to prevent higher than normal concentrations of EDTA and glycerol. The following is an example of a typical analytical single restriction enzyme digestion:
- 1. Under sterile conditions add the following components, in the order stated, to a sterile microcentrifuge tube.
| Sterile, nuclease-free water | 14µl |
| Restriction enzyme 10X buffer | 2µl |
| BSA, Acetylated (1mg/ml) | 2µl |
| DNA sample 0.2-1µg, in water or TE buffer | 1µl |
| Restriction enzyme, 2-10U | 1µl |
| Final volume | 20µl |
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- 2. Mix gently by pipetting. Centrifuge briefly at 12,000 x g in a micro centrifuge to collect the contents at the bottom of the tube.
- 3. Incubate at the optimum temperature for 1-4 hours.
- 4. Add 4µl of Blue/Orange 6X Loading Dye (or another appropriate DNA loading buffer), and proceed to gel analysis.
Larger scale restriction enzyme digestions can be accomplished by scaling this basic reaction proportionately.
Multiple Restriction Enzyme Digests
If all of the restriction enzymes in a multiple digest have the same optimal buffer, setting up the digest is straightforward. However, when this is not the case, several options are available.
- Use the optimal buffer supplied with one enzyme if the activity of the second enzyme is acceptable in that same buffer. Alternatively, acceptable activity for both enzymes may be achieved by using another of Promega’s 4-CORE® 10X Buffers. If one of the enzymes has less than 75% activity in the chosen buffer, the reaction time or the number of units of enzyme used may need to be increased. Be aware of possible star activity under non-optimal reaction conditions.
- Choose an isoschizomer or neoschizomer with more compatible buffer requirements.
- Perform a single digest with the first enzyme then inactivate that enzyme. Add the ingredients necessary for the second digest then add the second enzyme. For example, use a lower salt buffer and enzyme first, then inactivate the first enzyme, add enough salt to achieve the concentration required for the second digest, and add the second restriction enzyme.
Note: Perform each digest sequentially using the optimal buffers. This will require either a DNA precipitation or purification step after the first digest. Although this procedure involves more steps than those listed above, in situations where options 1-3 are not satisfactory, it may be the best alternative
