Cfrench:restriction1
9. ANALYTICAL RESTRICTION DIGESTS
To test the size or concentration of DNA or to determine the presence of restriction sites. For a single enzyme digest, mix in a 1.5 ml microcentrifuge tube:
- water 0 to 8 microlitres
- DNA 0 to 8 microlitres
- 10 x buffer 1 microlitre
- restriction enzyme 1 microlitre
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- total volume 10 microlitres
Mix by gently flicking the tube, then spin briefly (a few seconds) in the microcentrifuge to get all the liquid to the bottom of the tube. Incubate at 37˚C for 1 hour. Then add 2.5 microlitres loading buffer to the tube, mix and spin briefly, and load the entire 12.5 microlitres into one lane of a suitable electrophoresis gel.
The buffers we use are mainly from Promega. Enzymes from Promega have colour-coded lids to match the recommended buffer. For example, EcoRI and PstI have pink lids (recommended buffer H), XbaI has a yellow lid (buffer D) and SpeI has a brown lid (buffer B).
For plasmid DNA minipreps, generally use 6 microlitres of water and 2 microlitres of plasmid DNA unless you know from previous experience that the DNA is of higher or lower concentration than usual. Biobrick constructs in pSB1A2 often have a relatively low DNA concentration; try 2 microlitres, but if trying to see a small excised insert you may need to use 4 microlitres. pGemTe constructs usually have much more DNA, and 1 microlitre may well be sufficient.
For a double digest, use 0.5 microlitres of each enzyme. The total volume of restriction enzyme added should not exceed 10% of the total volume of the digestion mixture. You will need to choose a buffer that supports activity of both enzymes. There is a table in the appendix to the Promega catalogue which lists relative activity of restriction enzymes in different buffers. Here are suitable buffers for common biobrick-related double digests.
- EcoRI-PstI: buffer H
- EcoRI-SpeI: buffer E
- XbaI-PstI: buffer H
- SacI-SpeI: buffer E or multi-core buffer
- SacI-PstI: (no good buffer; avoid this combination)
- EcoRI-XbaI: buffer H
- SpeI-PstI: buffer B
When digesting multiple DNA samples with the same restriction enzymes (as, for example, when screening transformants) it is often easier to make up a single mixture and aliquot it into the digestion tubes. For example, to digest 6 DNA samples with Pst1 and EcoRI (as in screening for a biobrick insert), make the following mixture:
- water: 42 microlitres
- buffer H: 7 microlitres
- EcoRI: 3.5 microlitres
- PstI: 3.5 microlitres
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- total volume: 56 microlitres
Mix this and spin it briefly in a microcentrifuge. Transfer 8 microlitres to each of 6 tubes and add 2 microlitres plasmid DNA to each (if you know you will need more or less DNA than 2 microlitres, just adjust the amount of water in the mixture to compensate). Mix, spin briefly, and incubate. You will notice that the recipe above is enough for 7 digests rather than 6. If you try to make just exactly enough for 6, there will not be enough for all 6 tubes. Don’t ask me why, it is just a law of nature.
Safety notes: none of the solutions used in this procedure is significantly hazardous.
