Computer
Restriction Enzymes
and Lambda DNA
6B
Restriction enzymes have become an indispensable tool of molecular researchers over the past
fifty years. This unique group of enzymes function as molecular scissors when applied to nucleic
acids, in this case DNA. Forms of this special class of protein have been isolated from several
species of bacteria and are employed to make predictable, precise cuts in experimental DNA
samples. Currently, over two hundred different restriction enzymes are available to researchers,
each keying on a unique nucleotide recognition sequence. The mode of action of a restriction
enzyme is to attach and scan a strand of DNA looking for the presence of a specific nucleotide
sequence. When found, the DNA is cleaved at two opposite positions of the recognition site on
the sugar-phosphate backbone.
During this lab activity, prepared samples of bacteriophage lambda DNA (λ DNA) are used to
perform agarose gel electrophoresis. The samples result from λ DNA being digested with
different restriction enzymes. The individual digests of this bacteriophage, a 48,502 base pair
linear DNA segment, use two common restriction enzymes; EcoRI and HindIII. One of samples
is digested by EcoRI while another is cut by HindIII. There is a sample that is formed from a dual
digest that has both enzymes acting simultaneously on λ DNA. As a control, an uncut form of λ
DNA is also used.
The technique of agarose gel electrophoresis relies on an electric field being applied to a charged
gel matrix containing polar molecules. The response of these molecules to the electric field
induces them to migrate through the gel to the pole with an opposite charge. The rate of
molecular movement in a gel is determined by the charge, shape, structure and weight of the
molecule being studied. Negatively charged phosphate groups are present in DNA nucleotides
causing the molecule to migrate toward the positive end of the gel chamber. DNA fragments
maintain the same charge, shape, and structure, so base pair number differentiates the molecules
migration through the gel.
During this exercise, gel electrophoresis will be performed using the E-Gel Pre-Cast Agarose
Electrophoresis System with SYBR Safe stain. The Blue Digital Bioimaging System and
Logger Pro software will also be used to capture and analyze a digital photograph of your
electrophoresis results.
OBJECTIVES
In this experiment, you will
Perform agarose gel electrophoresis with the E-Gel System using four different samples of
λ DNA.
• Document and examine gel results with the Blue Digital Bioimaging System.
• Use Logger Pro to construct a standard curve and determine the base pair values from the
gel.
•
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6B - 1
Computer 6B
MATERIALS
computer
Logger Pro
Blue Digital Bioimaging System
ProScope HR
BlueView Transilluminator
1–10X lens
hood
stand
E-Gel Power Base & AC adapter
1.2% agarose E-Gel (SYBR Safe Stain)
2–20 µL pipettor
2–20 µL sterile pipettor tips
λ DNA samples
λ DNA – uncut
λ DNA – EcoRI digest
λ DNA – HindIII digest
λ DNA – EcoRI/HindIII digest
200 µL sterile water - ddH2O
lab mat
Nitrile gloves
microtube rack
ruler
waste container
PROCEDURE
Part I Perform Gel Electrophoresis
1. Prepare the E-Gel and the E-Gel Power Base.
a. Clean the lab table surface, wash your hands, glove, set the lab mat, and review lab safety
procedures.
b. Power the E-Gel Power Base with the AC adapter.
c. Remove the E-Gel from its packet and position it in the power base starting with the right
edge so the gel electrodes make contact with the power base electrodes. Press the E-Gel
down to lock it in place. A red light should go on and remain on at the top of the power
base.
d. (Pre-Run) Press and hold the 30-minute button on the power base until you hear the
double beep and the green light starts blinking. When you release the button, a required
two-minute warm-up cycle will begin. When the warm-up cycle is complete, the power
base will beep repeatedly. Press and release the same button to deactivate this warning. A
steady red light will appear.
e. The set of microtubes includes uncut λ DNA, λ DNA- EcoRI digest, λ DNA-HindIII
digest, λ DNA- EcoRI/HindIII digest, and ddH2O. Tap down each microtube. This action
maximizes the amount of solution at the bottom of each tube.
f. Remove the clear plastic comb from the top of the E-Gel and place it in the waste
container.
2. Load the E-Gel.
a. Twelve lanes are available in this E-Gel. Your instructor will suggest a loading sequence
for the λ DNA set. Write down your lane assignment information in Table 1.
b. Adjust the pipettor volume to 20 µL. Place a sterile tip on the pipettor, draw up the first
sample, load it in its designated well, and eject the tip into a waste container. Repeat this
step with each of the remaining three samples using a clean tip for each sample. Note:
Each well of the E-Gel requires a total volume of 20 µL. If there are blank wells, fill them
each with 20 µL of sterile water before running the gel.
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Advanced Biology with Vernier
Restriction Enzymes and Lambda DNA
3. Run the E-Gel.
a. Once all the wells are loaded, press the 30-minute button on the E-Gel power base to start
the electrophoresis run. The red light on the E-Gel power base should turn green to
indicate the run has begun.
b. While the E-Gel is running, clean up your work area by returning your materials to the
designated storage places. Dispose of nitrile gloves and wash your hands.
c. At the conclusion of the thirty-minute gel run, the E-Gel power base will beep repeatedly
and the light will flash red. Press one of the buttons to stop the beeping, disconnect the
power cable from the power base, and remove the E-Gel.
4. Use a ruler to measure, in millimeters, the distance across the top of E-Gel from the start of
the first well to the end of the last well. This distance will be used in Step 12. Record this
value in Table 2.
Part II Photodocumentation of Results
5. Start Logger Pro and choose New from the File menu.
6. Prepare the E-Gel and the BlueView Transilluminator.
a. Transfer the E-Gel to the central portion of the blue platform of the BlueView
Transilluminator. The top region of the E-Gel should be next to the hinge of the orange
lid.
b. Connect the BlueView Transilluminator to AC power and turn it on.
7. Position the ProScope.
a. Connect the 1–10X lens to the ProScope.
b. Connect the ProScope to the USB port.
c. Mount the ProScope to the stand and position the stand next to the transilluminator,
opposite the side with the hinge.
d. Level the ProScope so that its lens is
parallel to the surface of the
transilluminator.
8. Prepare Logger Pro for use.
a. Choose Gel Analysis ► Take Photo from
the Insert menu.
b. Orient and focus the ProScope so both the
bands and lane numbers are clear and
sharp. Note: Adjusting brightness to a
lower value under camera settings is often
helpful.
Figure 1
9. Place the Imaging Hood over the ProScope and the BlueView Transilluminator. Reach
through the flap of the hood to make final adjustments for best position, focus, and
resolution.
10. Once satisfied with the image, click
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. The screen should now resemble Figure 1.
6B - 3
Computer 6B
Part III Gel Analysis
The buttons along the right side of the gel photograph are used during gel analysis. The first four
are the primary Gel Analysis tools. Text above the photograph serves as a reminder of the next
step in the analysis.
11. Indicate the position of the wells on the photograph.
a. Click Set Origin, .
b. Click the photograph just to the left of the first well. A yellow coordinate system will
appear on the photograph.
c. Position the x-axis directly along the bottom edge of the wells. You can move the origin
by clicking either axis and dragging it to the desired location. The axis can be rotated by
clicking the round handle on the x-axis.
12. Convert the units of distance measured from pixel count into millimeters or centimeters.
a. Click Set Scale, .
b. Click and drag between the start of the first well and the end of the last well.
.
c. Enter the distance value from Table 2, including units. Click
13. Identify the bands and base pair values of the standard ladder using the λ DNA/EcoRI digest
lane as the standard ladder.
a.
b.
c.
d.
e.
Click Set Standard Ladder, .
Click the leading edge of the first band in the λ DNA/EcoRI digest lane.
Enter the number of base pairs for this band using the values in Table 3. Click
Click the next band in this lane and enter the base pair value. Click
.
Repeat this process for each visible band of the standard ladder. Logger Pro will
automatically create a standard curve on the graph.
.
14. Identity the experimental bands in the
remaining lanes. Logger Pro will plot bands,
record distance migrated and calculate the
respective number of base pairs.
a. Click Add Lane, , and choose Add
Lane.
b. Click the leading edge of the first band in
the first experimental lane. Notice that
when you click, three things happen: a
marker with a distinct shape and color is
placed on the photograph, a matching
marker is placed on the standard curve of
Figure 2
the graph, and the distance and number of
base pairs are added to the data table (see
Figure 2).
c. Click the leading edge of the next band in this lane.
d. Continue this process for each visible band in the experimental lane.
15. Repeat Step 14 for each remaining experimental lane.
16. Record the base pair values for the experimental lanes in Table 4. Not all cells will be filled.
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Advanced Biology with Vernier
Restriction Enzymes and Lambda DNA
17. (optional) Print the results of the E-Gel analysis.
DATA
Table 1
Lane assignments
Lane
Volume
λ DNA (µL)
λ DNA form used
Table 2
E-Gel scaling sistance
Distance across wells
mm
Table 3
Standard ladder values or λ DNA – EcoRI digest
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Band
Base pairs
1
21,226
2
7,421
3
5,904
4
5,643
5
4,878
6
3,530
6B - 5
Computer 6B
Table 4
Results
λ DNA – HindIII
Band
Base pairs
λ DNA – EcoRI/HindIII
Band
Base pairs
λ DNA
Band
1
1
1
2
2
2
3
3
3
4
4
4
5
5
5
6
6
6
7
7
7
8
8
8
9
9
9
10
10
10
Base pairs
QUESTIONS
1. Which restriction enzyme produced the most DNA fragments during the digest of λ DNA?
2. The gel used in this activity was 1.2% agarose. If the concentration of agarose were 2%, what
effect would this have on the migration of DNA fragments? What effect would a 0.8%
agarose gel have on this activity? What benefit would exist in using a higher concentration
agarose gel?
3. DNA molecules consist of two complimentary chains of nucleotides arranged in an antiparallel fashion to form a helical structure. This double stranded molecule contains paired
nucleotides following Chargoff’s Rule. Each nucleotide position along a segment of DNA
can be one of four forms; adenine, thymine, guanine, or cytosine. Considering the six base
pair recognition sequence for the restriction enzyme EcoRI, how often would you expect this
sequence to appear?
5’ …GAATTC… 3’
3’ …CTTAAG… 5’
Recognition sequence for Eco RI
Assuming your research project deals with an organism whose genome contained 12 million
base pairs. You have isolated and cleaned up a sample of the organisms DNA and want to
make a collection of smaller segments that you will use for the next stage of your research.
How many segments would you expect to result from a digest with EcoRI?
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Advanced Biology with Vernier
Restriction Enzymes and Lambda DNA
4. As observed from the results of your gel run, smaller fragments appear further from the well
than larger segments. Explain why the shorter DNA segments migrate furthest during agarose
gel electrophoresis.
5. Why is a semi-logarithmic graph used when creating a standard curve to determine base pair
lengths of experimental DNA segments?
6. What affect would reducing the voltage to your gel by twenty percent have on the observed
results?
7. Restriction enzymes make one of two types of cuts in DNA being digested. Some of these
enzymes produce sticky ends at the cut site while others produce blunt cuts. What are sticky
ends and what makes them important to recombinant DNA studies?
8. During a restriction digest using HindIII on DNA isolated from an insect, gel electrophoresis
was performed using a 1.0 % agarose gel. The results showed several bands with the same
light intensity. One band, however, was several times brighter than the others. Further
investigation in the literature revealed this brightness was due to a triplet of closely related
DNA segments. What could you do to better resolve these three bands in your gel?
Considering the activity just performed, how might you expand it to resolve a base pair count
for each of the bands?
9. Restriction enzymes can protect bacteria from many viral infections by attacking foreign
DNA and cutting it up into useless segments. What would stop a restriction enzyme from
digesting the DNA of the host bacteria that created it?
10. A process called restriction fragment length polymorphism (RFLP) has been used to initially
assess relatedness of different species. Explain how this process works. Discuss its
limitations.
EXTENSION
1. Restriction enzymes can be used with smaller DNA vectors, specifically plasmids, to give
much more controlled results. Using plasmid X, devise a map of this circular structure based
on the following restriction digest results. The uncut plasmid is known to consist of
4361 base pairs. Three restriction enzymes were used to characterize the plasmid through a
series of dual digests; enzymes A, B, and C. Results were run on an agarose gel and the
banding patterns were analyzed. Digest with enzymes A and B yielded two segments whose
lengths were 377 and 3984 base pairs, respectively. The second digest using enzymes A and
C produced a segment 748 base pairs long and another of 3613 base pairs. The final digest
using enzymes B and C resulted in two segments, one 1125 base pairs and the other with
3236 base pairs.
2. Using commercially available plasmids and restriction enzymes, e.g., pUC 19 and BsaI,
EcoRI, and HindIII, digest a plasmid with three separate dual digests. Use a 1.2% agarose gel
to separate the digest results. A low range DNA ladder will need to be used as a standard and
the results can be analyzed with Gel Analysis. From your results, reconstruct the plasmid and
its restriction sites.
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6B - 7
Experiment
TEACHER INFORMATION
6B
Restriction Enzymes
and Lambda DNA
1. This experiment correlates with Lab 6: Exercise 6B in the 2001 College Board’s AP Biology
Lab Manual. It requires the use of a computer and Logger Pro.
2. The student pages can be found on the CD that accompanies this book. See Appendix A for
more information.
3. This experiment, using the E-Gel® System, requires one 45-minute lab period to complete the
electrophoresis and documentation portions. Additional time may be needed to perform the
analysis portion. The experiment is designed for use with 1.2% agarose E-Gels stained with
SYBR Safe DNA gel stain.
4. Safety precautions strongly recommend that students wash hands before and after lab, wear
goggles, lab coat or apron, and protective gloves. Spent DNA microtubes and pipette tips
need to be collected and disposed of as Biohazardous material in a manner acceptable to local
health and sanitation codes. E-Gels stained with SYBR® Safe DNA gel stain need to be
disposed of in a similar manner.
5. This experiment is written for use with the E-Gel Pre-Cast Agarose System but can also be
done using standard gel electrophoresis equipment. When using standard gel electrophoresis
equipment with the BlueView Transilluminator, you can use SYBR Safe stain purchased
from Vernier or one of several other fluorescing stains. Stains with an excitation wavelength
in the blue range of 450 to 520 nm range will usually work. For use with nucleic acids, these
include but are not limited to SYBR Safe, SYBR® Gold, SYBR® Green I, GelGreen™, and
GelStar®. Several fluorescent stains for proteins are also available that work well with blue
light illumination.
6. When using E-Gels, each lane, whether used or left blank requires the addition of 20 µL of
fluid. Fill blank lanes with distilled water. Avoid over-filling, as this is a source of cross
contamination between lanes. Use one pipette tip per sample when loading the gel.
7. λ DNA samples can be obtained directly from several common suppliers including WARD’S
Natural Science. See Appendix G for more information. Obtain the following conventional
lambda DNA markers: Lambda DNA/EcoRI, Lambda DNA/HindIII, and Lambda
DNA/EcoRI + HindIII. Note: If 6X-loading dye has not been added to these markers, add at a
rate of one volume of 6X Loading Dye to five volumes of DNA solution and mix thoroughly.
Uncut Lambda DNA can also be obtained through the same suppliers. This material will
require dilution with a buffer (TAE or TBE) and 6X loading dye to a level comparable with
the markers.
Note: Methylated DNA with work for this activity but will not allow restriction digesting if it
is to be used for subsequent digest analysis.
8. A serial dilution trial of the nucleic acid concentration can be instructive when preparing this
lab. Using too much DNA in a lane will result in smeared bands that are difficult to analyze.
If using an E-Gel, use no more than 0.5 to 2.0 µg of total DNA per lane. The amount for
standard gels will vary depending on the width of the gel wells. If the concentration of the
lambda ladder is 0.5 µg/µL, 1–2 µL should be sufficient DNA for the 20µL sample used in
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6B - 1 T
Experiment 6B
one lane. DNA can be diluted by adding 1X buffer or ddH2O. If using a loading stain or dye,
remember that several of the DNA bands will possibly travel at a comparable rate to the
marking dye which may mask the presence of these DNA bands. Adding 1–2 µL of a 60%
glycerol solution will assist in keeping the DNA weighted down in the well and avoid
seepage.
To calculate the amount of DNA present in samples, multiply the concentration by the
volume. (µg/ µL) (µL) = µg.
To calculate concentration after dilution, remember C1V1 = C2V2.
9. You may want to have multiple groups use the same E-Gel by allocating lanes 1–4 to one
group, lanes 5–8 to another and lanes 9–12 to a third.
10. Make sure the correct power supply is used with the correct device. Using the wrong power
supply on the BlueView Transilluminator or the E-Gel power base could cause damage to the
instruments.
11. The BlueView Transilluminator will not function with the lid raised. It needs to be closed for
the magnetic safety switch to allow current to pass through the unit.
in procedure Step 10,
12. If the Logger Pro screen is not auto-arranging after clicking
check the Close Window and Auto Arrange boxes in the Take Gel Photo dialog box.
13. For the best photo of the gel banding patterns, you may want to adjust the camera settings
before your click
. From the Take Gel Photo dialog box, click the Camera Settings
button and go to Adjustments. Position the brightness slider somewhere between 10 and
25%. Be sure to have the dimmer switch of the BlueView Transilluminator turned to full
brightness and the Imaging Hood placed over the system. Continue to adjust until the best
results are obtained.
14. When performing a gel analysis, the sequence of events described in the procedure must be
followed in order. You cannot go back and change anything with a few exceptions. Points
placed on the photograph identifying experimental bands can be moved or deleted. Simply
click on the Select Point button, , then click on the point to be edited. Drag it with the
cursor to move it or use the Delete key on your keyboard to delete. Points for experimental
bands can be added by clicking on the Add Lane button, selecting the appropriate lane, then
clicking on the band in the photograph.
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Advanced Biology with Vernier
Restriction Enzymes and Lambda DNA
SAMPLE RESULTS
Table 1
Lane assignments
Lane
Volume
λ DNA (µL)
λ DNA form used
1
20µL
xxxx
2
20µL
xxxx
3
20µL
xxxx
4
20µL
xxxx
Table 2
E-Gel scaling distance
Distance across wells
xxxx
Table 3
Standard ladder values
For λ DNA – EcoRI digest
Band
Base pairs
1
21,226
2
7,421
3
5,904
4
5,643
5
4,878
6
3,530
The bands made by segments 3 and 4 will appear as a single band or doublet due to their
closeness.
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6B - 3 T
Experiment 6B
Table 4
Results
λ DNA – HindIII
λ DNA – EcoRI / HindIII
λ DNA
Band
Base pairs
Band
Base pairs
Band
Base pairs
1
xxxx
1
xxxx
1
xxxx
2
xxxx
2
xxxx
2
3
xxxx
3
xxxx
3
4
xxxx
4
xxxx
4
5
xxxx
5
xxxx
5
6
xxxx
6
xxxx
6
7
7
xxxx
7
8
8
xxxx
8
9
9
xxxx
9
10
10
xxxx
10
The values listed are ideal, student results will vary due to many factors that include
technique, sample concentrations, photograph band results, band positions in gel analysis
presence or absence of specific bands, etc.
ANSWERS TO QUESTIONS
Answers have been removed from the online versions of Vernier
curricular material in order to prevent inappropriate student use.
Graphs and data tables have also been obscured. Full answers and
sample data are available in the print versions of these labs.
6B - 4 T
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