BIOTECH Project: Restriction Digests of DNA
University of Arizona
Name:
Date:
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?
Why would we want to do this?
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?
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BIOTECH Project: Restriction Digests of DNA
University of Arizona
Analyzing Your Restriction Digest Data
Materials
DNA from restriction enzyme digests (BE SURE TO ADD 5 MICROLITERS LOADING DYE!!)
agarose
Tris-acetate/EDTA solution (TAE)
micropipette/tips
electrophoresis apparatus
Procedure:
1. Get your electrophoresis apparatus. Make sure the comb is in place (there should be 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?
3. While the agarose is hardening add 5 l of loading dye to each of your samples. What is the purpose of the
dye?
4. When the agarose gel is hard, take out the comb and stoppers.
5. Load your RESTRICTION DIGEST SAMPLES WITH LOADING DYE 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.
Draw a picture of your gel and indicate which samples are in which wells before you add DNA to the gel.
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?
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?
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?
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?
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
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Actual Bp Sequence
BIOTECH Project: Restriction Digests of DNA
University of Arizona
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