Restriction Endonuclease Digestion of a Plasmid

Margaret M. Dooley

Biology Department
College of Staten Island
The City University of New York
2800 Victory Boulevard, Staten Island, NY 10314
dooley@[Link]

Abstract: Recombinant DNA technology is widely utilized and therefore, undergraduate laboratory
courses incorporate these techniques. This reliable laboratory exercise introduces the student to
plasmid vectors, restriction enzymes, and agarose gel electrophoresis. Restriction digestion of
pBR325 with PstI and HindIII, in single and double digests, was performed and analyzed on an
agarose gel. Students constructed a standard curve based on the migration of fragments from a
HindIII digest of bacteriophage λ DNA and used that curve to estimate the sizes of DNA fragments
in the pBR325 digests. In this exercise, measurements on the largest λ fragment were included to
illustrate the resolution limits of agarose gels and the proper use of standard curves.

Introduction

The plasmid cloning vector, pBR325, carries genes coding resistance to the antibiotics
tetracycline, ampicillin and chloramphenicol. Unique restriction sites are located in each antibiotic
resistance gene, for example, PstI in ampicillin, BamHI in tetracycline and EcoRI in
chloramphenicol (Bolivar, 1978; Prentki et al., 1981) and at other positions around the plasmid, e.g.,
HindIII which is located in the promoter for the tetracycline resistance gene (Rodriguez et al., 1979).
The location of unique restriction sites at various positions around the plasmid permits the formation
of easily visualized and well separated restriction fragments. Thus, this vector is well suited for use
in instructional restriction analysis protocols. Using this vector also introduces the student to the
structure and utilization of the widely utilized plasmid cloning vector pBR322 and its derivatives.
Students digest pBR325 with HindIII and PstI, perform agarose gel electrophoresis, and estimate the
fragment sizes from a standard curve generated from a λ HindIII digest. In this exercise, this plot
includes the 23,130 base pair (bp) λ fragment to illustrate the limited resolution range of agarose gels
and the necessity of having points on the standard curve bracket the experimental measurements.

Materials and Methods

Materials and Solutions

Restriction enzymes, restriction buffers, and λ DNA were from New England BioLabs;
pBR325 was from Sigma-Aldrich. Plasmid DNA was resuspended in sterile TE buffer to 0.1 µg/µL,
and λ DNA to 0.4 µg/µL. Electrophoresis reagents (agarose, loading dye, Tris-Borate-EDTA (TBE)
buffer, ethidium bromide solution) and Tris-EDTA buffer can be purchased from Carolina
Biological Supply Company or prepared according to Sambrook et al. (1989). Tris-EDTA (TE) is
390 ABLE 2007 Proceedings Vol. 29

10mM Tris-HCl (pH 8.0), 1mM EDTA (pH 8.0); 5X TBE stock solution is 54 g Tris base, 27.5 g
boric acid, 20 ml 0.5 M EDTA (pH8.0); loading dye is 0.25% bromophenol blue, 0,25% xylene
cyanol FF, 40% (w/v) sucrose in water (Sambrook et al., 1989).

Safety
Students were carefully supervised when they started the electrophoresis to prevent any
safety hazards. Staining was with 1 µg/mL ethidium bromide which is a mutagen and suspect
carcinogen. Therefore, to minimize risk, students were not allowed to handle the staining solution,
aerosols were avoided, and a 5 mg/mL stock solution was purchased, not a powder. Ethidium
bromide solution was chemically inactivated before disposal (Quillardet and Hofnung, 1988) or
collected on commercially available filters which were discarded with hazardous waste. To protect
the retina and skin from shorter wavelength ultraviolet light, an ultraviolet absorbing shield was
always used when viewing gels.

Experimental Procedure
Students added all reagents as listed in Table 1 to digest plasmid (0.5 µg) with HindIII and
PstI in both single and double digests. A HindIII digest of λ DNA (2 µg) was also performed to
provide molecular weight standards on the agarose gel. After incubation for 1 hour at 37°C, samples
were heated to 60°C for 3 minutes to melt the λ cohesive ends before loading on a 0.8% agarose gel
and performing electrophoresis at 80-100 volts for approximately 1 hour until the dye front was 1-2
cm from the end of the gel. If the experiment could not be completed in one laboratory session,
samples were frozen after the restriction digestion and the 60°C incubation and electrophoresis was
performed during a second session. The technician stained the gels in 1 µg/mL ethidium bromide for
at least 1 hour and rinsed with water before viewing on an ultraviolet emitting transilluminator. The
gels were photographed with Polaroid 667 film. The fragment migration distances were measured on
an 8 X 11 inch print of the digitized photograph or alternatively, an expanded copy, or the Polaroid
print itself.

Table 1. Restriction reaction mixes

Tube # Water Buffer DNA Enzyme
1 10 µL 4 µL 5X HindIII buffer 5 µL λ 1 µL HindIII
2 10 µL 4 µL 5X PstI buffer 5 µL plasmid 1 µL PstI
3 9 µL 4 µL 5X HindIII buffer 5 µL plasmid 1 µL PstI AND 1 µL
HindIII
4 10 µL 4 µL 5X HindIII buffer 5 µL plasmid 1 µL HindIII
5 11 µL 4 µL 5X HindIII buffer 5 µL plasmid ---

Results and Discussion

The results of an ideal gel are shown in Figure 1A. Comparison of either single digest with
the λ ladder confirms the size of pBR325 which has 5,995 base pairs (bp). The double digest
 Poster Session 391

produces fragments that are approximately 2,400 and 3,600 bp as expected. Clearly, HindIII and PstI
each cleave pBR325 only once and at different sites on the plasmid. Most undigested plasmid
migrates closer to the wells than the linear pBR325 in the single digest lanes, confirming that both
restriction enzymes cleaved pBR325 and emphasizing the need to compare like conformations in
size determinations.

1A

1B

Figure 1A. Agarose gel electrophoresis of restriction digests. Lane 1, λ DNA digested with
HindIII (the size of each fragment is indicated in base pairs); lane 2, pBR325 digested with
PstI; lane 3, pBR325 digested with PstI and HindIII; lane 4, pBR325 digested with HindIII;
and lane 5, undigested pBR325. 1B. Standard curve for fragment size determination. The
distance each λ HindIII fragment migrated was measured and plotted (arithmetic X-axis)
verses the size in base pairs (logarithmic Y-axis).
392 ABLE 2007 Proceedings Vol. 29

Figure 1B shows the standard curve semilog plot that was used to determine the fragment
sizes. A 0.8% gel cannot resolve fragments in the 23,130 bp range and therefore, this λ fragment is
not on the linear portion of the plot which extends from 2,027 to at most 9,416 bp. This can be used
to illustrate the importance of bracketing the unknown points with the standard curve as a linear
relationship might not continue beyond the measured range. In the case of the λ HindIII ladder, if the
2,027-9,416 bp linear portion of the curve were incorrectly extrapolated beyond the highest point,
the apparent size of the 23,130 bp fragment would be 12,000 bp, nearly a two fold error. Clearly, it is
inappropriate to extrapolate a standard curve beyond the measured range.
The 2,027-9,416 bp linear portion of the standard curve also reflects the fragment sizes that
can be properly resolved; the steeper segment shows the size where the gel can no longer suitably
separate fragments. The lack of resolution of larger DNA’s can be emphasized by asking students to
predict how far 23,000 and 22,700 bp fragments would migrate (both 62 mm) compared to the easily
resolved 2,322 and 2,027 bp fragments (110 and 115 mm).
Assessment was from grading of student lab reports and a quiz. In this exercise, students
gained knowledge of the pBR325 cloning vehicle, the restriction digestion procedure, the agarose
gel electrophoresis procedure, the interpretation of agarose gel patterns, and certain dilution
procedures. The resolution limits of agarose gels and the correct use of standard curves were
emphasized by considering the data on the largest λ fragment. In addition, students acquired the
information and practice required to properly use micropipettors, microcentrifuges, and
electrophoresis equipment.

Literature Cited

Bolivar, F. 1978. Construction and Characterization of new cloning vehicles. Gene 4:121-136.
Prenrki, P., F. Karch, S. Iida and J. Meyer. 1981. The plasmid cloning vector pBR325 contains a
482 base pair long inverted duplication. Gene 14:289-299.
Quillardet, P. and M. Hofnung. 1988. Ethidium bromide and safety-Readers suggest alternative
solutions. (Letter to editor) Trends in Genetics 4: 89.
Rodriguez, R. L., R. W. West, H. L. Heyneker, F. Bolivar, H. W. Boyer. 1979. Characterizing wild-
type and mutant promoters of the tetracycline resistance gene in pBR313. Nucleic Acids
Research 6:3267-87.
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning A Laboratory Manual. Book
3. Second Edition. Cold Spring Harbor Laboratory Press, 1659 pages.

About the Author

Margaret M. Dooley earned her B.A. from Cornell University and her Ph.D. from Syracuse
University. She received postdoctoral training at the University of California, Davis and Rutgers
University before joining the faculty of the College of Staten Island where she teaches genetics,
microbiology and general biology courses.

©2007 Margaret M. Dooley