BIOTECH Project: Restriction Digests of DNA
University of Arizona
TEACHER GUIDE: Manipulating DNA and Genetic Engineering
Restriction Digests of DNA
How can we take genes from one organism and connect them with genes from another organism?
Using restriction enzymes, we can cut DNA from each organism and then ligate it together (e.g. the
ampicillin resistance gene and the luciferase gene into the plasmid backbone).
Why would we want to do this?
This is extremely useful if you want to have an organism make a protein it doesn't normally make (e.g.
bacteria making insulin). If we take the human insulin gene, cut it out of the human genome using
restriction enzymes, ligate it into a plasmid with the ampicillin resistance gene, and transform bacteria with
this plasmid, then all the ampicillin resistant bacteria will be making insulin that can be used to treat
diabetes.
Restriction digest plan
You will be digesting your DNA with two different enzymes. In one tube you will use HindIII and the other
EcoRI.
Step 1: Label your tubes with your initials and the enzyme you’re using (H for HindIII and E for EcoRI).
Step 2: Pipette 10 µl water into the bottom of each tube.
Please check off each component as you add it to your reaction tube. You can add these in any order.
Lambda DNA
Add 6 microliters (µl) of DNA to each tube.
Done
Buffer (Why do we add buffer?)
Add 2 microliters (µl) of buffer to your EcoRI tube.
Add 2 microliters (µl) of buffer to your HindIII tube.
Done
Enzyme
Add 2 microliters (µl) of the EcoRI enzyme to your EcoRI reaction tube.
Add 2 microliters (µl) of the HindIII enzyme to your HindIII reaction tube.
Done
Why do you only add 2 µl of buffer?
When you’ve visited all the stations, put your tube into the 37°C heat block or water bath. Why do we put the
reactions at 37°C?
Restriction enzymes are isolated from bacteria. In fact, they serve as a bacterium's immune system by
cutting up foreign DNA (like viral DNA). The environment of the bacteria is typically 37°C, which means
that these enzymes have evolved to work best at 37°C.
INCUBATE THE TUBES FOR AT LEAST 1 HOUR (can go overnight).
STUDENTS MUST ADD 5 MICROLITERS LOADING DYE TO EACH OF THEIR RESTRICTION
DIGESTS PRIOR TO LOADING THEIR GELS.
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BIOTECH Project: Restriction Digests of DNA
University of Arizona
Analyzing Your Restriction Digest Data
Materials
DNA from restriction enzyme digests
molecular weight markers
agarose
Tris-acetate/EDTA solution (TAE)
micropipette/tips
electrophoresis apparatus
Procedure:
1. Get your electrophoresis apparatus. Make sure the comb is in place (one comb near the black electrode) and
that there are stoppers at both ends of the gel space.
2. Pour hot agarose into the gel space until it reaches the top of the gel box. Let the agarose harden, which should
take about
10 minutes. Don’t touch/move your gel until it’s hard. Why not? If the agarose moves while it's hardening,
it will harden unevenly, making it more difficult for the DNA to move through evenly.
3. While the agarose is hardening add 5 l of loading dye to each of your samples. What is the purpose of the
dye? The dyes allow us to visualize the electrophoresis even though we cannot see the DNA. The dyes will
separate into two distinct bands and when the smallest band has migrated about ¾ of the way through the
gel, you can stop the gel.
4. When the agarose gel is hard, gently remove the stoppers and comb
5. Load your RESTRICTION DIGEST SAMPLES into the wells near the BLACK ELECTRODE. Why
near the black electrode? Be sure to keep track of which samples you loaded in which lanes. DNA is
negatively charged, so to move the DNA into the gel with electricity, the DNA needs to be loaded on the
negative or black side, it will then move towards the red. If it's loaded near the red electrode, it will
migrate off the gel.
Draw a picture of your gel and label which samples are where before you add DNA to the gel.
The samples are indistinguishable once they are loaded, so everyone needs to have a drawing of what they
will load where before they load their DNA samples. This drawing will be useful during the analysis, once
the gels are stained.
6. After you have added your samples pour TAE solution over your gel so that is it completely covered plus a
little more. What do you think the TAE solution is for? What do you think the TAE solution is for? TAE is
like saltwater - it conducts electricity, plus it is a buffer to control for pH changes
7. Run that gel!! Plug the electrodes into your gel box (red to red, black to black), being careful not to bump
your gel too much. Plug the power source set between 100 and 125 V into an outlet. How can you tell your gel
is running? It bubbles at the electrodes. This is a redox reaction, forming H2 gas at the black electrode and
O2 gas at the positive electrode.
After about 30 minutes the DNA should be sufficiently separated to analyze, the purple dye will have migrated
approximately 2/3 of the gel, turn off the power and carefully remove the gel. The gel is very fragile, take care to
not break it. You can remove the tray that you poured agarose on to and gently slide the gel into the staining tray.
At this point you cannot see the DNA, what can you see and how do the four different lanes compare? Draw a
picture of what you see.
Once you have placed your gel into the staining tray bring it to the staining station. Completely cover the gel
with methylene blue and cover try with saran wrap. Stain overnight.
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BIOTECH Project: Restriction Digests of DNA
University of Arizona
Next day--Viewing the gel: Discard the methylene and carefully place the gel onto a white light box. The gel is
very fragile so take care to not break it. Draw a picture of your stained gel, be as accurate as possible in drawing
the bands:
How many cuts did each restriction enzyme make? With Methylene blue staining usually only 6 bands of DNA will
be visible with Hindlll, though one of those bands (6,557) is difficult to see, because it blends in with the band (4,361)
closest to it. EcoR1 is actually 6 bands, but most students will only be able to distringuish 4, maybe 5. They will need
to determine which bands they are not able to visualize and give possibilities as to why.
Analysis of Restriction Digests of DNA:
Table 1
Hind III
Actual Base Pairing (Bp) Sequence
23,130
9,416
6,557
4,361
2,322
2,027
564 (may not be detected)
125 (may not be detected)
Measured Distance (mm)
Based on the DNA fragments you see with the HindII cut, how many nucleotides is the uncut DNA? Does this confirm
with the literature on lambda DNA? Lambda DNA is 48,502 bp in length. Obviously the students will not be able to
come up with that number based on their fragment sizes, see that they understand that some bands are difficult to
see due to the staining and the bands being close to each other in size. Have them speculate if they could do
something different and see all or more of the bands?
Graph your results for HindIII digest to determine the sizes of the EcoRI digest. Do those fragments add up to the size of
Lambda DNA? If not, provide possible explanation(s) as to why not.
Table 2
EcoRI
Measured Distance (mm)
Interpolated Bp Sequence
Band 1
Band 2
Band 3
Band 4
Band 5
Band 6
Band 7
Band 8
3
Actual Bp Sequence
21,226
7,421
5,804
5,643
4,878
3,530
BIOTECH Project: Restriction Digests of DNA
University of Arizona
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