Overview: Restriction enzymes are enzymes that digest DNAs at specific DNA sequences. In evolutionary studies, they can be used to determine if a DNA sequence recognized by a restriction enzyme varies between isolates. To digest DNA with a given restriction enzyme, DNA is combined with the enzyme in a buffer that is appropriate for optimal enzyme performance. The mixture is incubated for a period of time (from an hour to overnight, depending on the sample). The ability of the enzyme to digest the DNA is assayed by gel electrophoresis. Procedures vary with the type of DNA sample.
Materials and Supplies
- DNA samples
- Restriction enzymes
- 10X buffer for restriction enzymes
- Microtiter plates or 0.5 microcentrifuge tubes
- Oven, water bath or heating block set at 37oC
- Agarose (FMC Seakem LE)
- Electrophoresis gel units
- Power pack
- 10X tracking dye
- 5X TBE (Tris Borate EDTA Electrophoresis Buffer)
- Phi X DNA, HaeIII digest, 200 ng/10 m l
Restriction enzymes and appropriate buffers and temperatures: Each restriction enzyme has an optimal buffer and temperature for digestion of DNA. In order to get the best digestion activity, the correct buffer and temperature must be used. Several different commercial companies sell restriction enzymes (for example, Promega, Gibco). Usually, the appropriate buffer is shipped with purchase of the restriction enzyme. Buffers with the same number from different companies are not the same buffer. Promega and Gibco/BRL catalogs are excellent sources of information about the restriction enzymes that they sell including the correct conditions for digestion.
Pre-Digestion procedures: Two questions must be answered before DNAs are digested; 1) Which enzymes will digest your DNA fragment? 2) How many units of enzyme are required to digest a given amount of DNA in a given period of time?
Select one DNA sample to test different enzymes. The DNA that you have used as a PCR control is a good bet because you should have a large quantity of this fragment. Restriction enzymes are usually shipped from the company in a concentration of 10 units/m l. This concentration is usually greater than is needed to digest your DNA fragment but for your first tests, use this concentration. After you determine which enzymes cut your DNA fragment, you can try reducing the number of units required for each digestion.
a) Select the enzymes that you will use for your initial tests and determine which buffers to use.
b) Place the enzymes on ice. (Restriction enzymes should always be handled with gloves and kept cold.)
c) For each enzyme, set up the following reaction in a microtiter plate well or in a 0.5 ml microfuge tube.
|10X buffer (for this enzyme)||1 m l|
|Restriction enzyme||1 m l (10 units)|
|DNA||2-3 m l|
10 m l
The amount of DNA to be used in any reaction is a matter of judgement. For a good PCR reaction, 2-3 m l will usually provide sufficient DNA so that the digested fragments can be seen. Keep in mind that the digested fragments will be harder to see. They are smaller and will therefore bind less ethidium bromide. If more DNA is used in a reaction, there is the possibility that the PCR buffer will interfere with the digestion reaction but this varies from enzyme to enzyme.
2) Digestion (incubation of the DNA with the restriction enzyme)
a) If you are using a microtiter plate, seal the plate tightly with lab tape and place in the incubator at the correct temperature. Incubate for an hour. After incubation, allow reaction to cool, tap the tape to knock down any water droplets on the tape and then peel tape off. Add 1 m l of tracking dye and load on a gel.
b) If you are using microfuge tubes, close the cap and incubate for 1 hour. Spin the tubes for 10 seconds to bring condensate down. Add 1 m l tracking dye and load on a gel.
c) Run the gel, stain and observe. Record which enzymes have cut your DNA. You will use these enzymes in future experiments.
3) Determining how many units of an enzyme are necessary to cut your DNA:
a) Restriction enzymes are expensive and usually, only a single unit is required to digest a PCR product but restriction enzymes loose activity over time.
b) Select one restriction enzyme to test.
c) Prepare a 1X buffer using the correct buffer for that enzyme. Use 5 m L buffer and 45 m L sterile H2O to make 50 m L of 1X buffer.
d) Dilute the enzyme with 1X buffer as follows:
3 m L of enzyme (30 units) + 3 m L of sterile H2O = 30 units/6 m L = 5 units/m L
Mix thoroughly. (We’ll call this Solution A).
Take 3 m L of Solution A above (15 units) and add 3 m L of sterile H2O = 15 units/6 m L = 2.5 units/ m L. Mix thoroughly. (We’ll call this Solution B.)
Take 3 m L of Solution B above (7.5 units) and add 3 m L of sterile H2O = 7.5 units/6 m L = 1.25 units/ m L. Mix thoroughly. (We’ll call this Solution C)
e) Using your standard PCR product, set up the following reactions to determine the units of enzyme that will completely digest your DNA.
Ingredient Amount Amount Amount Amount H2O calculate calculate calculate calculate 10X buffer (for this enzyme) 1 m l 1 m l 1 m l 1 m l Restriction enzyme 1 m l (10 units) 1 m l of Soln(5 units) 1 m l of Soln B(2.5 units) 1 m L of Soln C(1.25 units) DNA 2-3 m l 2-3 m l 2-3 m l 2-3 m l TOTAL 10 m l 10 m l 10 m l 10 m l
f) Digest your DNA for 1 hour at the appropriate temperature in either a microtiter plate or in 0.5 ml microfuge tubes.
g) Run a gel as above
h) Examine the digestion products looking for the fewest units that digests your DNA completely. Use that level to digest all of your PCR products (see next section).
Digesting your PCR products from different collections:
You now know what enzymes will digest your DNA and how many units to use. Count the number of wells in the gel you plan to use. Reserve three wells, one at each end and one in the middle for molecular weight markers. Set up restriction digests for the remaining wells. You can make a master mix. An example for 27 samples is given below for 1 unit of enzyme/sample and 3 m L DNA:
Ingredient Amount per sample Master Mix (X27) H2O 5.9 m L 159.3 m L Dispense 7 m L of master mix to each tube or well 10X buffer (for this enzyme) 1 m L 27 m L Restriction enzyme 1 unit = 0.1 m L 27 units = 2.7 m L DNA 3 m L Add DNAs individually/well TOTAL 10 m L
The amount of 10X buffer and the total volume (bold text) are fixed. Everything else is determined experimentally.
Set up your reactions and digest for 1 hour at the appropriate temperature.
SOME RESTRICTION ENZYMES AND CONDITIONS FOR DIGESTION OF DNA
|Sma I||CCCGGG||4||30 C|
|Alu I||AGCT||B||1||37 C|
|Bam H1||GGATCC||E||3||37 C|
|Bgl I||GCCNNNNNGGC||2||37 C|
|Cla I||ATCGAT||1||37 C|
|Cfo I (Hha I)||GCGC||B||37 C|
|Dde I||CTNAG||D||37 C|
|Eco R1||GAATTC||H||3||37 C|
|Hae III||GGCC||2||37 C|
|Hind III||AAGCTT||E||2||37 C|
|Hinf I||GANTC||B||2||37 C|
|Hpa II (see Msp I)||CCGG||8||37 C|
|Kpn 1||GGTAAC||4||37 C|
|Hsp92 II||CATG||K||37 C|
|Mbo I||GATC||2||37 C|
|Msp I||CCGG||1||37 C|
|Pst I||CTGCAG||2||37 C|
|Pvu II||CAGCTG||6||37 C|
|Rsa I||GTAC||1||37 C|
|Sau 3A||GATC||4||37 C|
|Xba I||TCTAGA||2||37 C|
|Xho I||CTGGAG||D||2||37 C|
|Xmn I||GAANNNTTC||2||37 C|
|Bst X1||CCANNNNNNGC||2||55 C|
|Taq I||TCGA||2||65 C|