Diagnostic Restriction Digestion (DRD)
Updated 10/13/2020 12:09 PM
Introduction
Materials
- Plasmid DNA
- Restriction enzymes
- CutSmart Buffer
- MQ
- SNAPGENE software
- Ice to keep enzymes
- 1U cuts 1µg in 1h
Procedure
- Get as many tubes as there are plasmids to digest, label accordingly
- The last thing to do, since we don't want to destroy the enzymes: Get restriction enzymes from the freezer (keep on ice or in the blue frozen holder thing), and CutSmart Buffer. Be careful not to keep the enzymes outside of the freezer for too long, so immediately put them back in the freezer.
- Add to each tube the following:
DRD ingredients
A A | B B | C C | |
1 1 | Plasmid DNA | 5 μl (500ng) | |
2 2 | MQ | 3 μl | |
3 3 | CutSmart Buffer | 1 μl | |
4 4 | Enzyme | 0.5 μl each | |
5 5 | 10 μl total |
Note: check that you use enzymes that work at the same temperature as the incubating temperature (on snapgene)!
- To ensure that everything is properly mixed, the 1.5mL tubes can be spun down in the "normal" centrifuge by holding the "Pulse" button for 5 seconds. Do not vortex enzymes!
- Incubate at 37°C for 1 hour
- While the tubes are incubating, the gel can be prepared.
- Into each tube, add 2.5 μl loading dye
- Gel electrophoresis
DRD Snapgene
To find out if DigLig worked and the inserts are in plasmids correctly, we choose restriction enzymes for the plasmids so that one of the restriction sites lies on the insert and the other one accordingly on the backbone to produce unequal cuts. It is of course also possible to generate more than two fragments. That way, if the insert was inserted correctly, the digestion should produce distinguishable bands at different positions relative to the ladder.
- Simulated the Agarose gel in Snapgene that the diagnostic restriction digest should produce if the DRD worked:
Digest a plasmid with a set of enzymes to produce a gel that allows controlling if the insert is in the plasmid. Before starting with the actual samples search restriction sites that are cut by enzymes that are available in the lab and which cut in the insert and the backbone of your plasmid. How many cutting sites to choose can depend on the situation. The bands on the gel must be distinguishable (not less than 400bp short and more difference than around 500bp to be abel to distinguish them). Have a look at the Agarose Gel Simulation tool in SnapGene.