10th AP Biology Lab - Restriction Enzyme Cleavage of DNA & Electrophoresis - Part 1
EXPERIMENT OBJECTIVE:
The objective of this experiment is to develop an understanding of the role of restriction enzymes and agarose
gel electrophoresis to cut and size DNA.
LABORATORY SAFETY:
1. Goggles should be worn routinely as good laboratory practice.
2. Exercise extreme caution when working with equipment that is used in conjunction with the heating
and/or melting of reagents.
3. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS.
4. Exercise caution when using any electrical equipment in the laboratory.
5. Always wash hands thoroughly with soap and water after handling reagents or biological materials in the
laboratory.
LABORATORY NOTEBOOK RECORDINGS:
Address and record the following in your laboratory notebook.
Before starting the Experiment:
Write a hypothesis (If…then statement) that makes a prediction based on your expectations.
Reagents and Supplies
A – Bacteriophage Lambda DNA cut with Eco RI
B - Bacteriophage Lambda DNA cut with HindIII
C - Bacteriophage Lambda DNA – uncut (control)
UltraSpec-Agarose™ powder (form 0.8% solutions)
Concentrated electrophoresis buffer InstaStain®
Methylene Blue Methylene Blue PLUS™
Practice Gel Loading Solution
Equipment and materials
10 – 100 µL Digital Micropipetter (to measure 20 µL)
Horizontal gel electrophoresis apparatus
D.C. power supply
Electronic Scale
Microwave, hot plate or burner
125 ml flasks or beakers
Hot gloves
Safety goggles
disposable laboratory gloves
Large weight boats (for gel destaining)
DNA visualization system (an overhead projector)
Distilled or deionized water
In the data section:
Be sure to include:
Visuals of the electrophoresis gel at the beginning and also at the end of the run (2 pics) – with labels
Quantitative – set up a table to identify which well had which DNA
Measurements: In another table record the distances the different bands moved
The widths of the individual bands themselves
The size of the gel itself
Qualitative – gel consistency, color of the gel – before staining/after staining/after 3 rinses, color of
DNA fragments,
Analysis Section:
Graph – include labels for title, x-axis, y-axis; units for x-axis, y-axis; plot the points, extrapolate to
determine base pair sizes of the unknown DNA fragments; tell what the graph allows you to
determine – using specifics.
Questions
Background:
Restriction
Enzyme
Bgi I
Bam HI
Organism
Bacillus globigii
Bacillus amyloliquefaciens H
The discovery of restriction enzymes began a new era of
molecular genetics. These enzymes cut DNA in a highly
specific and reproducible way. This, in turn, made
molecular cloning, DNA mapping, sequencing and
various genome projects possible.
Restriction enzymes are endonucleases that catalyze
cleavage of phosphodiester bonds within both strands of
Eco RII
Escherichia coli, strain R 245
DNA. They require Mg+2 for activity and generate a 5
Hae III
Haemophilus aegyptius
prime (5') phosphate and a 3 prime (3') hydroxyl group at
the point of cleavage. The distinguishing feature of
Hind III
Haemophilus influenzae Rd
restriction enzymes is that they only cut DNA at very
specific base sequences. Restriction enzymes are
Figure 1
produced by many different species of bacteria (including
blue-green algae). Over 2,000 restriction enzymes have been discovered and characterized. More recently,
intron-coded yeast mitochondrial endonucleases have been discovered that also cut DNA. The recognition
sequences for these enzymes yield very few cuts in DNA and promise to be important new biological reagents
for DNA analysis.
Eco RI
Escherichia coli, strain RY 13
Restriction enzymes are named according to the organism from which they are isolated. This is done by using
the first letter of the genus followed by the first two letters of the species. Only certain strains or sub-strains of a
particular species may produce restriction enzymes. The type of strain or substrain sometimes follows the
species designation in the name. Finally, a Roman numeral is always used to designate one out of possibly
several different restriction enzymes produced by the same organism or by different substrains of the same
strain.
A restriction enzyme requires a specific double-stranded recognition sequence of nucleotide bases to cut DNA.
Recognition sites are usually 4 to 8 base pairs in length. Cleavage occurs within or near specific enzyme
recognition sites. The cleavage positions are indicated by arrows. Recognition sites are frequently symmetrical,
i.e., both DNA strands in the site have the same base sequence when read 5' to 3'. Such sequences are called
palindromes. Consider the recognition site and cleavage pattern of fco RI as an example.
5'-GAATTC-3'
3'-CTTAAG-5'
5'-G
AATTC-3'
3-CTTAA
G-5'
As shown above, Eco RI causes staggered cleavage
of its site. The resulting ends of the DNA fragments
are called "sticky" or "cohesive" ends.
Procedure:
A. Prepare Agarose Gel:
1. Prepare a 0.8% agarose concentration.
The concentration of agarose determines the size range of linear DNA fragments which
can be separated and resolved.
Done in
advance
% agarose (w/v)
0.6
0.7
1.0
1.2
1.5
Range of separation
(sizes in bases)
1000 – 20,000
800 – 12,000
500 – 10,000
400 - 6,000
200 – 3,000
2. Calculate gel volume.
Multiply the length (cm) x width (cm) x thickness (cm) of the desired gel
Length of tray = 9.3 cm , width of tray = 7.1 cm, desire thickness = 5 mm (careful note units),
3. Prepare the buffer composition.
The electrophoresis buffer conducts the electric field to and through the gel, and helps
maintain a balanced pH in the chamber.
To make 1X TBE Buffer (from 10X TBE Buffer) –
100 ml 10X TBE Buffer
900 ml distilled water
Mix thoroughly until clear. Store at room temperature. It can be reused several times.
Start
Here
4. Prepare the agarose solution: (For one 4 – 5 mm thick 0.8% agarose mini gel…)
(Teacher could add 5 g of agarpse tp 625 mL of 1x TBE buffer in advance or…)
a. Dissolve 0.24 g agarose in 30 ml of 1x TBE buffer in a 50 ml flask.
b. Stir well to suspend agarose.
c. Cover loosely and carefully heat – do not allow it to boil over!
1. Microwave: 1 to 1 ½ minutes for each flask in the microwave
2. Boiling water bath; 10–20 min. occasionally swirling until solution is clear
3. Heat directly on top of a hot plate or bunsen burner.
d. Allow the solution to cool to 60˚C (until you can comfortably hold the flask)
e. Add 2 drops of Blue Gel and Buffer Stain
B. Assemble and prepare the electrophoresis tray:
1. Place the tray on a level surface.
2. Slide the gates all the way up and snug the screws – alternating a little at a time on each screw.
3. Insert the 12-toothed comb into the tray (there is a notch and it only fits in level, one way)
4. Use distilled water to test for leakage. (slide the tray to a new area on the table and look for
signs of water leaking out from any of the edges. Snug up the screw(s) on the side that
leaks, slide it to a new, dry location and check again.) DO NOT over tighten the screws.
5. Remove the comb, pour out the distilled water.
6. Insert the comb into the tray again.
7. Pour the 60˚C agarose gel you prepared into the tray with a smooth motion avoiding air
bubbles. (If there are air bubbles you can break or remove them with a micropipettor.)
The gel should cover ½ the height of the comb.
8. Allow the gel to solidify (15 - 20 minutes – it will be translucent, slightly cloudy and firm)
9. Then carefully remove the comb by pulling it straight up and out of the wells it created.
10. Slightly loosen the screws just enough to push the gates down to expose the ends of the gel.
At this point the gel could be used right away or wrapped in saran wrap & stored in the refig for 1 week.
C. Set up the electrophoresis chamber:
1. Carefully insert the tray into the electrophoresis chamber, with the taller side on the same side
as the chamber’s electrical posts.
2. The wells created by the comb should be near the negative (black) pole end of the chamber.
3. Add approximately 150 ml of 1X TBE Buffer (Tris-borate-EDTA buffer) to the
electrophoresis chamber.
4. The buffer should slightly cover the gel, with the gel and wells submerged in buffer solution.
Do NOT fill the chamber above the electrode posts.
D. Load your DNA samples into the wells:
1. Centrifuge the DNA tubes (three at one time – evenly spaced) to pool all DNA to the
bottom of each tube – so it can be pipetted out..
2. Determine the load volume depending on the comb used and the thickness of the gel.
For a 5 mm thick gel, refer to the following chart:
Comb Well
Load Volume
6
40 µl
8
30 µl
10
25 µl
12
20 µl
3. Set the micropipettor to 20 µL (that’s the amount this kit gives). Ideally more like 35 µL
4. With a new pipette tip, extract the determined volume from DNA from sample tube A.
5. Refer to the diagram below: carefully submerge the tip into the first well, then load.
6. Continue steps 3 and 4 until you have loaded all three DNA samples as listed below:
Lane
1
2
3
Tube
A
B
C
DNA samples
Lambda/Eco RI
Lambda/ HindIII
Lambda DNA (uncut)
E. Running the gel:
1. After the DNA samples are loaded, slide and carefully snap the cover into place.
2. Connect the cathode (positive, red) plug from the gel box to the cathode (positive, red) input
port on the power supply.
3. Connect the anode (negative, black) plug from the gel box to the anode (negative, black)
input port on the power supply.
4. Plug in the power supply unit.
5. Turn the power supply unit on.
6. Set the unit to 150 volts.
7. Check to see that the current is flowing properly – you should see bubbles forming from the
two platinum electrodes.
8. Run the gel for a minimum of 30 minutes to a maximum of 1 hour.
At 15 minutes, check to see where the DNA fragments are – do not let them run off the
gel, turn off the unit before they do. Set a timer!
9. Unplug the power supply unit from the wall outlet.
10. Unplug the negative then positive probes from the power supply unit.
11. Remove the gel tray from the chamber.
12. Pour the buffer into the recycle beaker (it can be used again).
F. Staining the gel:
1. Gently slide the gel into a large weighing boat.
2. Pour ~ 50 mL of the Final DNA Stain over the gel and let soak for
5 to 10 minutes (if it appears very light, give it 5 more minutes.
OR
3. Using the “Instastain® Methylene Blue Card:”
a. Place the blue dye side of the card face down on top of the gel.
b. Firmly run your fingers several times over the entire surface of
the Instastain® card
c. To ensure continuous contact between the gel and the stain
card, place another weighing boat on top of the card, with
a weight, such as a small empty beaker.
d. Allow the Instastain® Methylene Blue to sit on the gel for
5 to 10 minutes.
~ if the gel appears very light, wet the gel surface with buffer or distilled
water and place the Instastain® card back on the gel for an additional 5 minutes.
G. Destaining of DNA:
1. Transfer the gel to another large weighing boat.
2. Add approximately 100 ml of distilled water to cover the gel.
~ Room temperature water takes longer to destain.
~ You can accelerate destaining by heating the water to 37˚C
~ Do NOT exceed 37˚ C, it may soften the gel & cause it to break.
3. Repeat the destaining process by changing the distilled water as needed.
~ Do not exceed 3 water changes, bands may become too light.
H. Visualization of DNA:
The larger DNA bands will initially be visible as dark blue bands against a lighter blue
background.
When the gel is completely destained, the larger DNA bands will become sharper and the smaller
bands will become visible. With additional destaining, the entire background will become
uniformly light blue.
I. Reading the gel:
1. The table below gives the actual size in base pairs (Act. bp) of lambda DNA fragments
generated by a HindIII digest:
2. Using your gel or the ideal gel shown in Figure 1, carefully measure the distance (in mm) each
HindIII and EcoRI fragment migrated from the origin (the well). Measure from the front edge
of the well to the front edge of each band. Enter the distances into the table above.
3. Match the base-pair sizes of the HindIII fragments with bands that appear in the ideal digest.
Label each band with its kilobase-pair (kbp) size. For example, 27,491 bp equals 27.5 kbp.
4. Set up the semilog paper with distance migrated as the x- (arithmetic) axis and the base-pair
length as the y- (logarithmic) axis. Then, plot the distance migrated versus the base-pair length
for each HindIII fragment.
5. Connect the data points with a best-fit line.
6. Locate on the x-axis the distance migrated by the first EcoRI fragment. Using a ruler, draw a
vertical line from this point to its intersection with a best-fit data line.
7. Extend a horizontal line from this point to the y-axis. This gives the base-pair size of this
EcoRI fragment.
8. Repeat steps 4f and 4g for each EcoRI fragment and place the answers under “Measured bp”
in the table above. After completion, your teacher will provide “Act. bp” data for comparison.
9. For which fragment sizes was your graph most accurate? For which fragment sizes was it least
accurate? What does this tell you about the resolving ability of agarose-gel electrophoresis?