Digestion

From 2007.igem.org

Latest revision as of 15:19, 20 September 2007

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 denatured DNA: 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.

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).

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.

Analyse for correct and complete digestion by gel electrophoresis using uncut material as a suitable control.