LAB 6:
Agarose Gel Electrophoresis of PCR Products
and Analysis of Results
I. Objectives
In today’s lab, you will practice preparing and running an agarose gel and learn how to interpret the
results of the PCR reactions they set up in Lab 5. By the end of today's lab, students should:

Feel fairly comfortable with setting up, loading, and running an agarose gel

Be able to interpret the results of the PCR reactions from Lab 5

Be able to explain which genes were targeted in the PCR reactions and why
II. Safety considerations

We will stain our gels with ethidium bromide to visualize the DNA as we did in the last lab.
Ethidium bromide is a powerful mutagen. To minimize exposure, all gel staining and photography will
be carried out at a separate station in the adjoining prep room in SQU 210. WEAR GLOVES during all
staining steps and be sure to discard these gloves before touching anything outside that area.
III. Introduction
A. MORE INFORMATION ABOUT THE PCR REACTIONS
1. E. COLI-SPECIFIC PRIMER SET
The primers we used to amplify DNA specifically from E. coli were developed by Hsu, C-H and Tsen, HY (International Journal of Microbiology, Vol 64, pp. 1-11, 2001). They target the E. coli form of the
malic acid dehydrogenase gene (mdh). The protein coded by this gene is an enzyme needed by
organisms that use the energy produced by the oxidation of nutrients to create ATP (oxidative
phosphorylation) In eukaryotes, oxidative phosphorylation takes place in the mitochondrion but in E.
coli it takes place in the plasma membrane (since bacteria have no membrane-bound internal
organelles). Although many organisms carry a gene coding for malic acid dehydrogenase, the primers
used in our experiment target the E. coli version of the gene, which is different in DNA sequence to
the genes coded by all other organisms.
BIO 2 Lab Manual, Fall 2009 Version 10/12/9
Lab 6, Page 1
The primers have the following sequences:
Emdh-1: 5' - ACTGAAAGGCAAACAGCCAAG - 3' (1123-1144)
Emdh2:
5' - CGTTCTGTTCAAATGGCCTCAGG - 3' (1514-1492)
The numbers after the primers refer to the nucleotides in the gene that the primer targets (e.g. the
"1123-1144" primer anneals to nucleotides 1123-1144 of the E. coli malic acid dehydrogenase gene
and will anneal to the 3' to 5' strand of the gene).
From this information, you can calculate the predicted PCR product size as 1514-1123 = 392 bp.
What annealing temperature would you choose for this reaction? Why?
2. UNIVERSAL BACTERIA PRIMER SET
The primers we used to amplify DNA from bacteria were developed by Lu, J-J, Perng, C-L, Yess, S-Y,
and Wan, C-C. (Journal of Clinical Microbiology, Vol 38, pp. 2076-2080, 2000). They target the 16S
rRNA gene, which is highly conserved among bacteria and codes for one of the RNA molecules that
forms part of the integral structure of the 30S ribosomal subunit of bacteria. Since eukaryotes do not
carry this gene and all (or at least most) bacteria do, a productive PCR reaction indicates the presence
of bacterial DNA.
The primers have the following sequences:
U1: 5' - CCAGCAGCCGCGGTAATACG - 3' (518-537)
U2: 5' - ATCGGCTACCTTGTTACGACTTC - 3' (1513-1491)
Calculate the expected PCR product size: __________bp
What annealing temperature would you choose for this reaction? Why?
3. COLIFORM SPECIFIC PRIMER SET
The coliform-specific primer set we used if from Xiang-Tao, M., Zhou, Y-P., Lei, H, and Deng, L.
(Enzyme and Microbial Technology, Vol 30(5), pp. 583-589, 2002). The primers target the Lac Z gene,
which codes for the enzyme beta-galactosidase. This enzyme breaks the bond between glucose and
galactose in lactose, enabling coliforms to utilize lactose as an energy source to make ATP.
ZR-2548-5’: 5’ – CACCATGCCGTGGGTTTCAATATT-3’ (2525-2548)
ZL-1675-5’: 5' - ATGAAAGCTGGCTACAGGAAGGCC- 3' (1675-1649)
BIO 2 Lab Manual, Fall 2009 Version 10/12/9
Lab 6, Page 2
Calculate the expected PCR product size: __________bp
What annealing temperature would you choose for this reaction? Why?
4.
PCR REACTION CONDITIONS
The PCR reactions you prepared last week contained the following:
38 uL Reaction Mix containing:
 5 uL 10X Mg-free taq buffer
 2 uL 25 mM MgCl2
 1 uL 10 mM dNTPs
 0.5 uL Taq polymerase
 29.5 uL nuclease-free water
10 uL
0.2 uM Primer Set (universal bacterial, E. coli-specific, or coliform-specific)
2 uL Template DNA
50 uL
TOTAL VOLUME
The thermocycler program was as follows:
Step 1: 94 deg C
Step 2:
Step 3:
Step 4:
5 minutes (initial denaturation)
94
oC
1 min (denaturation)
55
oC
45 sec (annealing)
72
oC
1 min (extension)
Step 5: Cycle steps 2-4, 29 more times
Step 6: 4 oC
HOLD
IV. Things to Do
PART A. AGAROSE GEL ELECTROPHORESIS OF PCR PRODUCTS
1.
Put on a pair of gloves. If you have long hair, tie it back.
2.
Pour an agarose gel, using the same technique as in Lab 6. Pour only one gel per group. (40
mL of agarose is needed to fill the casting tray.)
BIO 2 Lab Manual, Fall 2009 Version 10/12/9
Lab 6, Page 3
3.
While you are waiting for the gel to cool, retrieve your PCR reactions. Label 8 1.5-mL
microcentrifuge tubes (1-8) and add set up your samples for loading on the gel as follows:
Tube #
DNA
Sterile water
6X loading dye
1
20 uL reaction 1
--
5 uL
2
20 uL reaction 2
--
5 uL
3
20 uL reaction 3
--
5 uL
4
20 uL reaction 4
--
5 uL
5
20 uL reaction 5
--
5 uL
6
20 uL reaction 6
--
5 uL
7
20 uL reaction 7
--
5 uL
8
10 uL DNA ladder
--
--
*DNA ladder already contains loading dye; you do not need to add more.
4.
When your gel has cooled, carefully remove the comb (pulling straight up) and place the gel in
the gel rig. Then add enough 1X TAE running buffer to cover. Make sure that the wells are
filled with buffer and that the gel is well-covered by buffer.
5.
Load the samples onto the gel, loading from left to right in numerical order.
6.
When your samples are loaded, add the gel rig lid and connect the leads to the power supply.
Turn on the voltage to 100 V. Look for the appearance of bubbles on each end of the rig to
confirm that you have a current.
7.
Keep an eye on your gel, though, and watch as the dyes in the loading dye begin to separate
as they move through the gel at different rates.
8.
The gel should run for about 1 hour (until the first dye band is approximately ¾ of the way
down the gel). When the run is complete, disconnect the leads, remove the lid off the
apparatus. Carefully remove the casting tray (and gel) from the apparatus. Hold the gel on
either end so that it won't slip off the casting tray as you carry it to the staining station in SQU
210.
9.
Locate the area and transfer your gel into one of the clear staining trays—just slide it gently
off the casting tray. Wearing gloves, carefully submerge the gel in ethidium bromide
staining solution and let stand for 10 min.
10.
After the 10 min. staining period, carefully pour the ethidium solution back into its original
bottle. Submerge the gel in just enough tap water to cover the gel and let stand 5 min.
BIO 2 Lab Manual, Fall 2009 Version 10/12/9
Lab 6, Page 4
11.
Following the 5 minute destain, pour the rinse water into the bottle labeled ethidium bromide
rinse (not back into the ethidium bromide stock bottle).
12.
Transfer the gel onto the UV transilluminator in the digital imager chamber.
13.
Observe and capture an image of the gel according to the instructions posted there (or
described by your instructor).
14.
Print copies of the image for each member of your group.
15.
When you are done imaging your gel, discard the gel in the biohazard bag provided along with
your gloves. Again be careful not to touch anything that will leave this area while wearing
gloves used to handle ethidium bromide containing solutions.
16.
Tape your gel image into your notebook.
17.
Examine each lane of your gel. In your notebook record the expected outcome in each lane
and the observed outcome.
18.
Also, in your notebook, thoroughly describe what your observations allow you to conclude
about the identity of the DNA in each lane and the identity of the organism you isolated DNA
from.
V. Lab Clean-Up

Make sure your gel tray, comb, and gel apparatus have been rinsed with dI water and have
been placed back on your bench

Return your bacteria DNA extracts, unused loading dye, and unused PCR reagents to the front
bench.

Throw out all your other tubes in the trash.

GLOVES OR OTHER ITEMS THAT HAVE BEEN IN CONTACT WITH ETHIDIUM BROMIDE
(OTHER THAN THE GEL RIGS, CASTING TRAYS, AND COMBS) SHOULD BE DISPOSED
OF IN THE CONTAINER LABELED ETHIDIUM BROMIDE WASTE.
BIO 2 Lab Manual, Fall 2009 Version 10/12/9
Lab 6, Page 5