Download DNA Restriction and Electrophoresis

DNA Restriction and Electrophoresis
Agarose gel electrophoresis is a widely used technique for the analysis of nucleic acids and
proteins. The main purpose of this technique is to separate or fractionate molecules apart from
one another. This technique involves moving negatively charged nucleic acid molecules through
an agarose matrix with an electric field (electrophoresis). Shorter molecules move faster and
migrate further than longer ones
Background:
Agarose, which is extracted from seaweed, is a linear polysaccharide consisting of the repeating
modified galactose units. An agarose gel is created by suspending dry agarose in a buffer
solution, boiling until the solution becomes clear, pouring it into a casting tray and allowing it to
cool. The result is a flexible gelatin-like slab containing a matrix that acts like a molecular sieve.
During electrophoresis, the gel is submersed in a chamber containing a buffer solution and a
positive and negative electrode. The DNA to be analyzed is forced through the pores of the gel
by the electrical current. Under an electrical field, DNA will move to the positive electrode and
away from the negative electrode. Several factors influence how fast the DNA moves, including;
the strength of the electrical field, the concentration of agarose in the gel and most importantly,
the size of the DNA molecules. Smaller DNA molecules move through the agarose faster than
larger molecules. DNA itself is not visible within an agarose gel. The DNA will be visualized
by the use of a dye that binds to DNA.
Restriction Enzymes
Bacteriophages are viruses that infect bacteria by injecting their own DNA into the bacterial cell
and force the bacteria to multiply the viral DNA. Bacteria have responded by evolving a natural
defense, called restriction enzymes, to cut up and destroy the invading DNA. These enzymes
search the viral DNA for specific palindromic sequences of base pairs, such as (GAATTCs), and
cut up the DNA into pieces at these sites. The actual place in the palindrome where the DNA is
cut is called a restriction site. An important feature of restriction enzymes is that each enzyme
only recognizes a specific palindrome and cuts the DNA only at that specific sequence of bases.
A palindrome can be repeated a number of times on a strand of DNA, and the specific restriction
enzymes will cut all those palindromes at their restriction sites.
Procedure Day One:
Restriction Digest Preparation:
1. Label four 1.5mL microcentrifuge tubes as follows:
B = BamHI digest
E = EcoRI digest
H = HindIII digest
C = Control
2. Pipet the reagents into each tube according to the table below (Use a fresh tip for each
transfer):
Tube DNA
B
4l
E
4l
H
4l
C
4l
Buffer BamHI EcoRI HindIII
5l
1l
5l
1l
5l
1l
5l
Water
1l
3. Mix the components by gently flicking the tube with your finger and tapping gently on the
table to collect liquid to the tube bottom. Pulse-spin in a microcentrifuge to collect all
the liquid to the bottom of the tube.
4. Incubate the tubes for 1 hour at 37°C.
Agarose Gel Preparation (0.8% agarose gel):
1. Weigh out 0.8g of agarose.
2. Add 100ml 1X TBE Buffer and 20l SYBR Safe stain to the agarose.
3. Using the hot plate and a stir bar, bring the agarose solution to a boil.
*Be careful to NOT allow the solution to boil over!
4. Prepare gel cast and well comb.
5. Once agarose solution has cooled a bit, pour the solution into the gel cast. Allow the get to
solidify.
6. Once your gel has solidified label it, wrap it in Saran wrap and place it into the refrigerator
until the next lab period.
Procedure Day Two:
Prepare restriction digests for loading and sample loading:
1. Remove restriction digests from the freezer and allow them to thaw.
2. Remove your gel from the refrigerator, remove the Saran wrap and allow the gel to warm to
room temperature.
3. Once the digests have thawed, pulse-spin them in a microcentrifuge to collect all the liquid to
the bottom of the tube.
4. Add 3l of loading dye to each reaction tube.
5. Label a separate microcentrifuge tube “SM” (for size marker). To this tube add 10l HindIII,
10l dH2O and 3l loading dye.
6. Gently remove the comb, pulling it straight up and out of the set agarose. Place the gel along
with the casting tray in the electrophoresis chamber that the wells made by the comb are at the
negative electrode.
7. Fill the electrophoresis apparatus with 1X TBE buffer so that the entire gel is submerged.
8. Load the entire contents of each of your reactions into a separate well. Be sure to note the
order in which you loaded your samples.
9. Close the top of the electrophoresis apparatus, connect the electrical leads to the power supply.
10. Turn on the power supply and electrophorese for approximately 45 minutes at 150V.
11. Turn off the power supply, disconnect leads from inputs and remove the top of the
electrophoresis box.
12. Visualize and photograph your gel.