RESTRICTION MAPPING OF PLASMID DNA
Introduction
Restriction mapping is one of the first steps used to characterize novel DNA, especially
plasmids. Plasmids are small circular pieces of DNA found in bacteria that often contain antibiotic
resistant genes. During the process of restriction mapping, the DNA of interest is digested with various
restriction enzymes. The digested DNA is then loaded into the wells of an agarose gel and the
fragments are separated by size, where smaller fragments move faster than larger ones. Using the sizes
of the restriction fragments, a map of the DNA is created.
Restriction enzymes or restriction endonucleases are essential tools in recombinant DNA
methodology. Several hundred have been isolated from a variety of prokaryotic organisms. Restriction
enzymes recognize specific DNA sequences in double stranded DNA (usually a four to six base pair
sequence of nucleotides) and digest the DNA at these sites. The result is the production of fragments of
DNA of DNA of various lengths.
When any molecule enters an electric field, the mobility or speed at which it will move is
influenced by the charge of the molecule, the strength of the electrical field, the size and shape of the
molecule, and the density of the gel through which it is migrating. The lower the percentage gel, the
faster the DNA will move through. When all molecules are positioned at a uniform starting site on a
gel and a gel is placed in a chamber containing a buffer solution and electricity is applied, the
molecules will migrate and appear as bands. Nucleic acids, like DNA and RNA, move because of the
charged phosphate group in the backbone of the DNA molecule. Because the phosphates are
negatively charged at neutral pH, the DNA will migrate through the gel toward the positive electrode.
Procedure
1. Remove the tape and comb from the precast 0.8% agarose gel.
2. Place the gel in the electrophoresis chamber and cover the gel with buffer.
3. Load 20λ of the each predigested DNA sample into the wells of the agarose gel in the following
order:
a. λDNA/Pst I molecular weight markers
b. pMAP/Pst I
c. pMAP/Pst I/Hpa I
d. pMAP/Pst I/Ssp I
e. pMAP/Pst I/Hpa I/ Ssp I
4. Cover the chamber and run the gel at 120V for about 1 hr
5. After electrophoresis is complete, unplug the chamber, remove the gel and place it in the
staining tray. Cover the gel with stain for 15-20 minutes.
6. Destain the gel with dH2O using several changes of water.
7. View and photograph the gel.
8. Measure the distance in mm that the λDNA/Pst I molecular weight markers migrated in the gel
and record in Table I
9. Graph Table I on semilog paper to create a standard curve
10. Measure the distance the other fragments migrated in the gel and record in table II.
11. Using the standard curve, from Table I and the distances migrated from Table II, determine the
approximate sizes of the fragments from the other 4 digests and record them in Table III.
Table I: Distance λDNA/Pst I fragments migrated
Size of fragment in base pairs
11497
4700
2838
2450
2140
1986
1700
1159
1083
805
Distance migrated (mm)
Table II: distance pMAP fragments migrated (in mm)
Band
1
2
3
4
pMAP/Pst I
pMAP/Pst I/Hpa I
pMAP/Pst I/Ssp I
pMAP/Pst I/Hpa I/Ssp I
pMAP/Pst I/Ssp I
pMAP/Pst I/Hpa I/Ssp I
Table III: Size of the DNA fragments (base pairs)
Band
1
2
3
4
pMAP/Pst I
pMAP/Pst I/Hpa I
Analysis of Results
1. Determine the total size in base pairs of the pMAP plasmid.
2. How many Pst I sites are in the plasmid? How did you determine that? Hint: what is a plasmid?
3. Draw a map of the pMAP plasmid with the location of the Pst I sites and their approximate
sizes.
4. How many Ssp I sites are there in pMAP? How did you determine that?
5. Draw a map of pMAP with the positions of Pst I and Ssp I sites and their approximate sizes.
6. Why did you put the Ssp I site where you did?
7. How many Hpa I sites are there? How did you determine that?
8. Draw a map of pMAP with the positions of Pst I and Hpa I sites and their approximate sizes.
9. Why did you put the Hpa I site where you did?
10. Draw a map of pMAP with the Pst I, Ssp I and Hpa I sites and their approximate sizes.