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Lab 10: DNA Electrophoresis
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Introduction
You have already poured and run vertical gels for separation of proteins. During this lab,
you will work in groups of four to pour and run horizontal gels for size separation of
nucleic acids. You will then prepare and load samples of you RT-PCR products from Lab
9.
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Objectives
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To pour a horizontal agarose gel
To perform DNA electrophoresis in a horizontal agarose gel
To evaluate RT-PCR products from Lab 10 experiments
References
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Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual. Volume 1,
Section 6, “Gel Electrophoresis of DNA.”
Davis, Kuehl, and Battey, Basic Methods in Molecular Biology, 2nd ed. 1994.
Barker. At the Bench: a Laboratory Navigator.
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Reagents, Supplies, and Equipment
4.1
Reagents
1. 6X DNA Sample buffer
30% glycerol
0.25% (w/v) bromophenol blue
0.25% (w/v) xylene cyanol
3 mL glycerol
25 mg bromophenol blue
25 mg xylene cyanol
7 mL distilled H2O, store at -20°C
2. 5X TBE (Used at 0.5X dilution)
(5X)0.09 M Tris
(5X)0.09 M boric acid
(5X)2 mM EDTA
54 g Tris base
27.5 g boric acid
3.72 g Na2 EDTA٠2H2O
Add 900 mL distilled water, pH to 8.3. Add
additional distilled water to 1 L final volume.
3. SYBR Safe in 0.5X TBE Buffer – Invitrogen (s33110)
4. Agarose (Super Resolution) - Genessee (20-106)
5. 100 bp DNA Ladder – Invitrogen (15628019)
4.2
Supplies
1. Plastic Wrap
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4.3
Equipment
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Horizontal electrophoresis unit
Power Source
UV transilluminator
UV safety equipment (face shield, gloves)
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5
Protocol
Each gel will be poured, loaded, and run by groups of four.
5.1
Assembling the electrophoresis unit
1. Wash gel former, chamber, and combs with detergent, then rinse with deionized water.
2. Assemble the unit for casting the gel in the
assembly. Begin by placing the UV transparent gel
tray (small, open on two ends) into the middle of
electrophoresis bath with the electrode banana
plugs.
3. Insert the tapered baffles (small wedge shapes) on
each side of the UV transparent gel tray. This
should form a well, in which you will pour your gel.
The baffles should be pushed in firmly, to prevent
leaks, but not so tightly that they can’t be easily removed.
4. Test for leakage using distilled water.
5. Insert the gel comb. The comb should enter the gel
approximately 1 cm from the side of the gel closest
to the black connector.
6. If the unit is facing you with the electrical
connections in the back, that means that the comb
should be inserted on the left side of the gel. These
electrophoresis units have definite black and red
sides, and putting the comb on the wrong side will
mean having to switch things around later.
7. Using the white knobs on the comb, adjust the
comb length until the tip of the comb is ~2 mm
above the bottom of the gel (the comb
shouldn’t touch the plastic under the gel). The
easiest way to do this is to place two glass
slides, one atop the other, on the bottom of the
well, and then use that to set your comb height.
5.2
~2 mm
~1 cm
Pouring the gel
1. Add 0.32 g of agarose to 40 mL of SYBR Safe in 0.5X TBE (for a 0.8% gel). Microwave
to dissolve agarose (continue heating and swirling gently until solution is clear), then let
cool to ~60°C (hand hot).
8. Pour the gel into the well formed by the UV transparent gel tray and the tapered baffles.
Add gel until the gel level is at least 5 mm higher than the bottom of the comb (pretty
much filling the tray), and set the leftover gel aside. Wait 15 minutes or until gelled, using
your leftovers to gauge the gelling time.
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9. Add enough 0.5X TBE buffer to keep your gel wet before use. Gently remove the comb,
placing your gloved thumb carefully in the middle of the gel if necessary – the gel will
slide around in its well otherwise.
10. Remove the baffles from each side. Now the gel can contact the buffer wells in the
electrophoresis cell, and you are ready to load samples on the gel and run it.
5.3
Loading and running the gel
1. Label 3 1.5 mL tubes as ‘Sample’, ‘No RT’, and ‘Control’.
2. Add 15 µL of each of these three RT-PCR products to 3 µL of DNA sample buffer in the
labeled tubes. Mix gently by sealing the cap and inverting the tube in your hand.
Centrifuge briefly to move the fluid back to the bottom. In a separate tube, add 2.5 µL of
DNA ladder to 0.5 µL of DNA sample buffer.
3. Heat sample tubes at 60°C for 5 minutes. Do not heat the DNA ladder! Let cool briefly
before loading. Again, centrifuge briefly to move the fluid to the bottom of the tube.
4. To the gel apparatus, add enough 0.5X TBE to fill both reservoirs and to overflow the
surface of the gel to a depth of 2-3 mm.
Blank
Control – 18 µL
No RT– 18 µL
Sample – 18 µL
DNA ladder (standard)
Control – 18 µL
No RT– 18 µL
Sample – 18 µL
5. Load your gel using the following picture as a guide. You and your partner will use one
side of the gel, reserving the other side for the other group. To the DNA ladder well, add
the entire 3 µL you’ve prepared.
* Use same loading volume as the ‘Sample’ well to the left
6. Run the gel for ~45-60 minutes at 100V, or until the two visible (blue & purple) dyes in the
sample buffer have sufficiently separated. When the leading dye front is nearly two-thirds
of the way into the gel, stop – the two dye fronts should divide the entire gel
approximately into thirds.
5.5
Staining and photographing the gel
1. Place plastic wrap over UV light. Place gel and tray atop the plastic wrap.
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2. Visualize the DNA with UV light. Wear face shield and gloves to protect yourself from the
UV!
3. Photograph your gel using the appropriate filter for SYBR safe.
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Results & Discussion
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What did this experiment tell you about the RT-PCR experiment?
The last homework had us compare our primers with sequences in the BLAST
nucleotide database. What was the purpose of this? How could it help you to
understand your results today?
Why don’t we quantify the amount of DNA on this gel, as we did for the Western Blot,
using NIH-image? If we did quantify the DNA bands, what would it tell us?
Homework
Analyze the microarray data (available for download on the class webpage) using ScanAlyze.
Instructions for using the program are also available on the web page.
The green channel mRNA was collected from healthy primary cells in vivo. The red channel
mRNA was collected from a tissue engineered device seeded with the same kind of primary cells
and cultured for one month in vitro.
You should turn in the spreadsheet that ScanAlyze will generate and that you will then modify,
and answer the following questions:
1. What genes are significantly upregulated (doubled or more in expression) or
downregulated (halved or less in expression) when these cells are taken out of the body
and cultured in your device for one month? Identify the genes by row & column number.
2. Look carefully at your results. Some of your spots indicate negative expression.
Explain this result.
3. Take a second glance at your list for (1). Are there any genes you might question,
taking into consideration your thoughts for (2)? Why?
Please note: this homework is due the second Friday after the Microarray lecture.
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