Restriction enzyme digestion of DNA (basic method)

1. Restriction enzyme digestion of DNA(basic method) Kris-itd.unair.ac.id (for education purpose only

2. How much DNA to digest? The big question. You may be digesting your DNA just to look at it (an analytical gel) or to cut a band out of the gel for further treatment (a preparative gel). Either way you want to be able to see the DNA bands under UV light in an ethidium-bromide-stained gel. Typically, a band is easily visible if it contains about 20ng of DNA. Now consider an example. Suppose you are digesting a plasmid that comprises 3kb of vector and 2kb of insert. You are using EcoRI (a common restriction enzyme) and you expect to see three bands: the linearised vector (3kb), the 5' end of the insert (0.5kb) and the 3' end of the insert (1.5kb). In order to see the smallest band (0.5kb) you want it to contain at least 20ng of DNA. The smallest band is 1/10th the size of the uncut plasmid. Therefore you need to cut 10x20ng, that is 200ng of DNA (0.2µg). Then your three bands will contain 120ng, 20ng and 60ng of DNA respectively. All three bands will be clearly visible on the gel and the biggest band will be six times brighter than the smallest band.

3. Now imagine cutting the same plasmid with BamHI (another popular restriction enzyme) and that BamHI only cuts the plasmid once, to linearise it. If you digest 200ng of DNA in this case then the band will contain 200ng of DNA and will be very bright and will probably be overloaded. Too much DNA loaded onto a gel is a bad thing. The band appears to run fast (implying that it is smaller than it really is) and in extreme cases can mess up the electrical field for the other bands, making them appear the wrong size also. Too little DNA is only a problem in that you will not be able to see the smallest bands because they are too faint. Having said all that, I usually digest 2–4µL of the 50µL obtained from a kit miniprep. But you see how it depends on the number and size of the bands expected.

4. Typical protocol Combine the following in a microfuge tube in order; 1µl 10x Buffer 6.5µl H2O 2µl DNA 0.5µl Enzyme Buffer: This is the 10x buffer that comes with the restriction enzyme. Most companies have about 4 different kinds of buffer (called A,B,C,D or 1,2,3,4 etc.) and occasionally a "unique buffer" for a particular enzyme. Do not worry about this. Many restriction enzymes are forgiving about buffers. EcoRI, for example, works excellently in all four of the standard buffers. You can check in the enzyme company catalogue (NEB, Stratagene, Promega, Roche etc.) where there is a page devoted to enzyme/buffer compatibility. Fundamentally it is a question of salt concentration (high or low), Magnesium concentration (high or low) and pH.H2O: Standard distilled water. It is always good to add the buffer and water into the tube first. If you put the enzyme in straight on top of the buffer then it may become irreversibly denaturedDNA: As discussed above. Kit-prepared DNA is very good. If it is "home-made" miniprep DNA then salt can often be a problem. This can interfere with the enzyme (if it does not like salt) and can also make the gel run funny.Enzyme: 0.5µl of enzyme is plenty for a miniprep digestion. Do not use more enzyme than 10% of the final reaction volume (ie. not more than 1µl in this case). This is because the enzyme storage buffer contains antifreeze (glycerol) to allow it to survive at –20°C. The glycerol will inhibit the digestion if present in sufficient quantities. Incubate for 1 hour at 37°C in a waterbath. Meanwhile prepare the agarose gel.

5. Pre-mixes If you are doing many digests then it may be worthwhile to make up a pre-mix in order to save on pipetting. Suppose you are doing 16 digestions, all with the same enzyme, on 16 different DNA samples (but look at colony screening by PCR if appropriate). You should set up a 17x pre-mix (always do 1 extra to make up for slight pipetting inaccuracies) containing buffer, water, and enzyme. For the above example you would combine, in order, 17µl 10x buffer, 110.5µl H2O, and 8.5µl of enzyme. Mix by sucking up and down with the pipette on addition of the final ingredient (enzyme). Avoid air-bubbles when mixing because the enzyme will get trapped at the air/liquid interface and become denatured. Place 2µl of DNA into each of 16 microfuge tubes then add 8µl of pre-mix on top, mixing by sucking up and down a few times. Use a fresh pipette tip for each tube. Double digests You may be digesting your DNA with two (or more) enzymes. This is fine but you have to make sure to use the buffer that will be most compatible with all the enzymes. There is usually a page in the back of an enzyme catalogue devoted to this problem. Rule of thumb: popular old-fashioned enzymes (EcoRI, BamHI) are the best and most forgiving, weird modern restriction enzymes (Xyz3A-1) are often most fastidious. Also, don't forget the 10% volume rule (about minimizing glycerol).

6. Special conditions A few enzymes require special conditions. It will say in the catalogue. Some require BSA (bovine serum albumin) added into the mixture. This is usually provided free with the enzyme at 100x concentration. Some require weak detergents (eg. triton-X-100) to reduce surface tension. Some require to be incubated at temperatures other than 37°C (e.g. 50°C). If you have not heard of the enzyme before then it is worth checking for these things in the catalogue. Some manufacturers require all kinds of weird stuff when the enzyme works perfectly fine without it. If you cannot obtain some arcane ingredient then don't panic. It will probably work perfectly well without it. Star activity This is when the enzyme cuts at sites other than its cognate element. eg. EcoRI is supposed to only cut GAATTC but, under extreme conditions, it might possibly cut CAATTC also. Extreme conditions is the operative phrase here. I have done many 1000s of restriction digests and have never experienced trouble due to star activity. Links Enzyme manufacters provide lots of information about restriction digestion. Also lots of really good basic tutorial stuff. This is available both in the back of the catalogue (look at NEB, Stratagene and Invitrogen) and on the web; www.neb.comwww.stratagene.comwww.promega.comwww.invitrogen.comwww.roche.com