Cloning and Sequencing Explorer Series

BACKGROUND
CHAPTER 3: ELECTROPHORESIS

CHAPTER 3
Background
Agarose Gel Electrophoresis
Agarose gel electrophoresis separates DNA fragments by size. PCR products or other DNA
fragments are loaded into an agarose gel slab, which is in a chamber filled with a
conductive buffer solution. A direct current is passed between wire electrodes at each end
of the chamber. Since DNA fragments are negatively charged, they will be drawn toward
the positive pole (anode) when placed in an electric field. The matrix of the agarose gel acts
as a molecular sieve through which smaller DNA fragments can move more easily than
larger ones. Therefore, the rate at which a DNA fragment migrates through the gel is
inversely proportional to its size in base pairs (bp). Over a period of time, smaller DNA
fragments will travel farther than larger ones. Fragments of the same size stay together and
migrate as single bands of DNA.
The sizes of DNA fragments are determined through comparison with a molecular weight
standard — in the case of this lab, a molecular weight ruler that has a series of bands
differing in size by 500 bp increments from 500 bp up to 5,000 bp. The expected sizes of
the target GAPDH PCR products range from 500 to 2,500 bp, depending on the plant
species chosen.
Agarose gel electrophoresis is a useful tool since it allows determination of the number of
PCR products (useful since multiple GAPDH genes may be amplified using this protocol),
the size of these PCR products, the success of the PCR (the presence and intensity of the
DNA bands), whether there has been contamination of the sample based on examination of
the negative control, and whether primer-dimers have been amplified.

(–)

(+)

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Protocol
Overview
The products of both the initial
and nested PCR reactions will be
analyzed by agarose gel
electrophoresis to assess PCR
success; the number of amplified
bands and their sizes will be
examined to evaluate the success
of each round of PCR.
Cloning the GAPC gene
• Identify and extract gDNA from plants
• Amplify region of GAPC gene using PCR
• Assess the results of PCR
• Purify the PCR product
• Ligate PCR product into a plasmid vector
• Transform bacteria with the plasmid

Safety Note: This kit includes • Isolate plasmid from the bacteria
ethidium bromide (CAS • Sequence DNA
#1239-45-8). Treat it as toxic. • Perform bioinformatics analysis of the cloned gene
Handle with care and follow
standard laboratory practices,
including wearing eye protection,
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gloves, and a laboratory coat to avoid contact with eyes, skin, and clothing. Please refer
to the Material Safety Data Sheet (MSDS) for complete safety information.
Safety Note: UV light is used to visualize ethidium bromide-stained DNA. UV light can
cause eye damage and burns. Do not look directly at the light, wear UV-protective eye
goggles, and avoid skin exposure.

Student Workstations
Each student team will require the following items to electrophorese their PCR samples:

Material Needed for Each Workstation Quantity

Horizontal electrophoresis chamber 1
Power supply 1
20 µl adjustable-volume micropipets and filter tips 1
Microcentrifuge tubes for preparing samples and aliquoting 10
500 bp molecular weight ruler 12 µl
5x Orange G loading dye 35 µl

Common Workstation

Material Required
Materials to cast agarose gels
Gel visualization and documentation system

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Preparation for Electrophoresis

1. Plan the gel electrophoresis and complete the table with the samples that will be run on
the gel.

Lane Sample Resulting Band

Number Sample Volume (µl) Sizes (bp)
1 500 bp molecular weight ruler 10 µl 500, 1,000, 1,500,
2,000, 2,500, 3,000,
3,500, 4,000, 4,500,
5,000

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2. Refer to Appendix A for detailed instructions on casting agarose gels, preparing
electrophoresis running buffer, and selecting electrophoresis conditions.
Cast a 1% agarose gel with the appropriate number of wells for analysis.
Prepare sufficient electrophoresis running buffer to run your samples. Note: Running buffer
can be reused 2–3 times.

Experimental Procedure for Electrophoresis

1. Label a microcentrifuge tube for each PCR sample.
2. Transfer 20 µl of each intial PCR reaction into a clean labeled microcentrifuge tube. Add
5 µl of loading dye into the tube. Mix up and down to mix. Note: Do not add loading dye
directly to the blue initial PCR tubes — the loading dye can inhibit the next round of PCR.
Loading dye is supplied as a 5x concentrate.
3. To assess the success of the nested PCR round, pipet 5 µl of each yellow nested PCR
into a microcentrifuge tube and mix it with 1 µl of 5x loading dye. A smaller volume of
the nested PCR is loaded because nested PCR is usually more efficient and produces
very intense bands that can obscure bands in adjacent wells if samples are overloaded.
4. Place a 1% agarose gel in the electrophoresis chamber. Pour electrophoresis running
buffer into the chamber until it just covers the gel by 1–2 mm.

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5. Load 10 µl of the 500 bp molecular weight ruler into the first well. The bands of the ruler
are 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500 and 5,000 bp. See
example gel in next section.
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6. Load 20 µl from each microcentrifuge tube containing an initial PCR with added loading
dye. Load 5 µl from each tube containing a nested PCR with added loading dye into
the wells of the gel according to your plan.
7. Connect your electrophoresis chamber to the power supply and turn on the power. If
you are using 1x TAE as the electrophoresis running buffer, run the gel at 100 V for 30
minutes. If you are using the fast gel protocol (Appendix A) with 0.25x TAE buffer, run
the gel at 200 V for 20 minutes.

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8. On completion of electrophoresis, visualize the DNA bands (using appropriate safety

equipment) and acquire an image of your gel according to your instructor's directions.
Paste image below and label it.

Results Analysis From the PCR

Construct a table of your results for each sample (including controls) that includes: the
sample name, whether a band or multiple bands are present, the size of the bands, and the
intensity of the bands relative to a particular band on the 500 bp molecular weight ruler.

Sample Name # of Bands Size of Band(s) Intensity of Band(s)

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The fragment of GAPC that has been targeted in this laboratory series varies in size
between plant species. The expected sizes of fragments from the initial round of PCR are
0.5 to 2.5 kb. The expected sizes of fragments from the second round of nested PCR are
slightly smaller than the size of the PCR product from the initial round. It is likely that
multiple DNA fragments of similar sizes will be amplified from the genomic DNA (gDNA) of
some plants, most likely due to amplification of multiple GAPC genes that are highly
homologous. For example, three bands are frequently amplified from the initial PCR of
Arabidopsis gDNA and two of these are amplified after nested PCR.

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An example of electrophoresis results of the initial and nested PCR. A 1% TAE agarose gel was loaded with initial (20 µl) or
nested (5 µl) PCR samples generated from specified genomic DNA extracted using the Nucleic Acid Extraction module or control DNA.
Lane 1- 500 bp molecular weight ruler (10 µl),
Lane 2- PCR of grass gDNA with initial primers (20 µl)
Lane 3- PCR of product from lane 2 with nested primers (5 µl)
Lane 4- PCR of thistle gDNA with initial primers (20 µl)
Lane 5- PCR of product from lane 4 with nested primers (5 µl)
Lane 6- PCR of control Arabidopsis gDNA with initial primers (20 µl)
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Lane 7- PCR of product from lane 6 with nested primers (5 µl)

Lane 8- PCR of pGAP plasmid control with initial primers (20 µl)
Lane 9- PCR of pGAP plasmid control with nested primers (5 µl)

What conclusions can you make from your PCR experiment? Did you obtain PCR products
of the expected sizes from your experimental plants?

Deciding Which Plant GAPC to Clone

Once all students in the class have their results, it is time to decide which plant will be used
for cloning. Although two plants were chosen for investigation, only a single plant’s GAPC
gene should be cloned. It is recommended that the plant chosen be the one that generated
the cleanest PCR product (fewest background bands), with good band intensity of an
appropriately sized fragment. It is acceptable to clone doublets since each linearized
plasmid is expected to ligate to a single DNA fragment. Be aware that cloning doublets may
result in colonies of transformed E. coli containing different gene sequences.
It is highly recommended that the entire class clone GAPC from the same plant, so that the
data obtained will be more reliable. Cloning the same gene multiple times will provide this
reliability, called depth of coverage, which will help to resolve any ambiguous base pairs
when the gene is sequenced. Remember that the sequencing data obtained from this lab
may provide new data for the scientific community at large; thus, it is vital the data provided
be as accurate as possible.
It is recommended that one or two student groups perform an additional PCR purification,
ligation, and transformation of the Arabidopsis GAPC PCR product, both as a positive
control for the class and as a backup in case ligations using PCR products from an
experimental plant are unsuccessful.
State which plant gene the class has decided to clone _______________________________
Why was this plant chosen?
______________________________________________________________________________

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Focus Questions for Electrophoresis

1. What is the purpose of the agarose gel?

2. What purpose does electrophoresis running buffer serve?

3. What purpose does the loading dye serve?

FOCUS QUESTIONS
4. What is the purpose of the molecular weight ruler?

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Electrophoresis – Quick Guide

1. Label a microcentrifuge tube for each PCR.

2. DO NOT add loading dye directly to blue initial

PCR reactions. In a separate tube add 5 µl
of 5x loading dye and add 20 µl of initial PCR
reaction and pipet up and down to mix.

3. Add 1 µl of 5x loading dye and 5 µl of yellow

nested PCR reaction to a separate tube and
pipet up and down to mix.

4. Place a 1% agarose gel in the electrophoresis

chamber. Pour electrophoresis buffer into the
(+)
chamber until it just covers the gel by 1–2 mm. (–)

5. Load 10 µl of the 500 bp molecular weight

ruler into lane one of the gel.

6. Refering to your electrophoresis plan, load 20 µl of

blue initial PCR reactions with loading dye and 5 µl of
the corrresponding yellow nested PCR reactions with
loading dye into the wells of a 1% gel.
QUICK GUIDE
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7. Connect the electrophoresis chamber to the

power supply and turn on the power. Run the gel
at 100V for 30 min unless otherwise instructed.

8. Upon completion of electrophoresis, visualize

your DNA fragments according to your instructor’s
directions.

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