Restriction Digest
Molecular Biology Lab #3
Background:
Restriction endonucleases are enzymes that have been isolated from bacteria. A large
number of these enzymes (over 3300 currently described) have been obtained from a
wide variety of bacterial types. Restriction endonucleases cleave within the DNA
molecule and at characteristic restriction sites for each specific enzyme. The restriction
site is usually composed of 4 to 8 base pairs that are organized in a specific sequence that
is recognized by the enzyme. Different restriction enzymes cleave in different patterns to
generate blunt or overhanging ends (sticky ends) of the DNA molecule. Many restriction
sites are composed of short inverted repeats.
Restriction enzymes are an extremely important tool in molecular biology. The restriction
enzyme sites within any DNA molecule are readily predicted from the sequence of bases.
Restriction digestion is used to separate fragments of genes for manipulation in the lab
(i.e. cloning or mutagenesis). Digestion with restriction endonucleases produces a
specific pattern of DNA bands when the reaction products are separated by gel
electrophoresis. This is termed the restriction pattern. The restriction pattern is diagnostic
of a specific DNA sequence, and it is frequently used to confirm the identity of specific
DNA fragments (for example, your unknown DNA).
Restriction endonucleases serve an important function in bacterial defense. Because they
are able to cleave any foreign DNA that enters the bacteria, they are capable of
inactivating intracellular bacterial pathogens. Interestingly, the bacteria also contain
specific methylases, enzymes that add a methyl group to the restriction site of enzymes
that the bacteria produce. This methylation prevents the restriction endonuclease from
digesting the bacterial DNA and killing the host cell.
The REBASE website http://rebase.neb.com/rebase/rebase.html is a very informative site
that contains everything that you would want to know about any restriction endonuclease.
You should navigate this site during incubation of your restriction digest and work on the
homework assignment.
Where can you get restriction enzymes? The easiest source is to purchase them from a
biotechnology company. They range in price from relatively cheap (BamHI = $30/2000
units) to very expensive (RsrII = $820/2000 units). The REBASE website links to many
biotechnology websites which makes purchasing or comparative shopping for restriction
enzymes easy.
How do you store restriction enzymes? They are usually stored dissolved in glycerol so
that they may be kept at -20º without actually freezing. You just pull the vial out of the
freezer and use it. Be careful about keeping the stock tube of enzyme cold. Place it on ice
water whenever you are not using it. This will preserve the enzyme activity. Don’t allow
the enzyme to remain at room temperature.
How much is enough? Usually 10 units of enzyme per microgram of recombinant DNA
is plenty. Digest at 37ºC for 1 to 24 hours. Digest longer if you use less enzyme or if you
are digesting cellular DNA (i.e. Southern blotting, later in the course).
Do all restriction endonucleases work under the same conditions? No, each enzyme
requires very specific conditions to work optimally. Usually, the amount of salt is very
important. When you buy an enzyme, a small vial of 10x buffer is usually supplied with
the enzyme. See the REBASE web site for an explanation of STAR activity.
What happens if you need to digest your DNA with 2 different restriction enzymes?
This occurs often when a fragment of DNA is cloned into two different restriction sites
(directional cloning). It is possible to do a double digest if the 2 enzymes are able to work
in the same buffer. This is easy. You just allow both enzymes to digest the DNA at the
same time. Sequential digests must be performed if the 2 enzymes cannot work under the
same conditions. Then you must digest with one enzyme, precipitate the DNA, resuspend
the DNA in an appropriate buffer, and digest with the second enzyme.
What can go wrong? Any contaminants in your DNA, such as agarose or phenol, can
inhibit the reaction. If the enzyme is left out at room temp for any length of time, it often
can lose activity.
Objectives: The purpose of this lab is to introduce you to restriction endonucleases and
provide practical experience setting up a restriction digest of plasmid DNA.
Materials:
37ºC and 60ºC water baths
restriction enzymes BamHI and EcoRI
React 3 buffer for the reaction
Micro pipetters and yellow tips
Eppendorf tubes
Deionized water to dilute DNA
TE buffer to resuspend precipitated DNA
BioRad spectrophotometer
Quartz cuvette
Microcentrifuge
Procedure:
1. If the DNA is stored as a precipitate, you should centrifuge for 5 min in a
microfuge to pellet the DNA. Resuspend the pellet in 50 to 200 l of 1x TE
buffer. TE buffer is a mixture of Tris buffer at pH 8 and EDTA. DNA is very
stable when stored in TE buffer. Mix carefully to ascertain that the DNA is
completely dissolved.
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2. Determine the concentration of DNA. Turn on the spectrophotometer and push
the DNA/RNA button and then the dsDNA button. Place 700 ml of 1x TE buffer
in the quartz cuvette and gently insert it into the cuvette holder of the spec. Be
gentle. The quartz cuvette and the instrument are expensive. Close the cover and
push the blank button to zero the instrument.
3. Dilute your DNA samples into 1x TE buffer for analysis. Add 5 l of DNA to
1000 l of 1x TE buffer in an eppendorf tube. Mix by shaking the tube. Pipette
the 700 l of DNA solution into the cuvette, insert cuvette into the
spectrophotometer, and close the lid. Push the sample button to record the
absorbance at 260 nm.
4. Read the concentration of DNA from the dial and multiply by your dilution factor
(x200) to calculate the actual concentration of your sample. A solution with a
value of 1 (A260 measurement) contains about 50 g of DNA / ml.
5. Touch the 260/280 button to calculate the ratio of absorbance at the 2
wavelengths. This value is usually in the range of 1.8 to 2.0 Very low readings
suggest contamination of your DNA with other molecules such as protein or
phenol. Record all information.
6. Set up your restriction digest in a total volume of 20 l. Add 2 g of each
recombinant DNA to a separate eppendorf tube. Add 2 l of the appropriate
restriction enzyme buffer to each tube. Use React3 for BamHI and EcoRI. Add
deionized water to each tube to bring the total volume to 19 l. Label eppendorf
tubes on the cap.
7. Add enzyme. Remove the tube of restriction enzyme from the ice bath and
carefully pipette 1 l into each of your reactions. Use a clean tip each time you
pipette from the stock tube of enzyme. This prevents contamination of enzyme
with your DNA sample. Keep the samples on ice and gently mix by pipetting up
and down with a yellow tip. Spin briefly (10 sec) in an eppendorf tube to collect
the reaction at the bottom of the tube.
8. Incubate the restriction digest reaction for 1 hour at 37ºC in a water bath. Place
each of your tubes in a small floating rack, close caps securely, and move the rack
to the water bath. Check periodically to see that the temperature remains at 37ºC
and that the caps of tubes remain closed.
9. After 1 hour, transfer your tubes to the 60ºC water bath to inactivate the enzyme.
Spin briefly in a microcentrifuge to collect the reaction in the bottom of the tube.
10. Store at -20ºC until next week. If you had sufficient time, you would proceed to
load your samples into a minigel to check the pattern of restriction fragments.
11. Clean up your work area!
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