6a Molecular Biology: Electrophoresis
In this laboratory, you will investigate some basic principles of genetic engineering. Plasmids containing specific fragments of foreign DNA will be used to transform Escherichia coli cells, conferring antibiotic
(ampicillin) resistance. Restriction enzyme digests of phage lambda DNA also will be used to demonstrate techniques for separating and identifying DNA fragments using gel electrophoresis.
In this experiment, three samples of DNA from the bacteriophage lambda (48,502 base pairs in length) are separated using gel electrophoresis: one cut with the restriction endonuclease EcoRI, one cut with the
restriction endonuclease HindIII, and one uncut control.
The DNA samples are loaded into wells of an agarose gel and electrophoresed. An electrical field applied across the gel causes the DNA fragments in the samples to move from their origins (sample wells) through the gel matrix toward the positive. Smaller DNA fragments migrate faster than the larger ones, so restriction fragments of differing sizes become concentrated into separate bands during electrophoresis. The characteristic number and pattern of bands produced by each restriction enzyme are, in effect, a “DNA fingerprint.” The restriction patterns are made visible by staining with the Carolina BLU；, which binds to DNA.
Figure 1 Figure 2
Restriction endonucleases recognize specific DNA sequences in the double-stranded DNA and digest the DNA at the sites. The result is the production of fragments of DNA of various lengths corresponding to the distance between identical DNA sequences within the chromosome. Some restriction enzymes cut cleanly through the DNA helix at the same position on both strands to produce fragments with blunt ends (Figure 1). Other endonucleses cleave each strand off-center at specific nucleotides to produce fragments with “overhangs” or sticky ends. By using the same restriction enzyme to “cut” DNA from two different organisms, complementary
“overhangs” or sticky ends will be produced and can realign in a “template complement” manner, allowing the DNA from two sources to be “recombined.”
By taking DNA fragments and systematically reinserting the fragments into an organism with minimal genetic material, it is possible to determine the function of particular gene sequences. In this way the genome or chromosomal character of the organism can be dissected rearranged, and tested for function and organization. Further, when a gene sequence of interest has been identified, it is possible to remove it from the chromosome and insert it into a plasmid that is capable of generating many copies of itself and the foreign gene sequence (Cloning).
Section A: Before doing this laboratory you should understand:
AP Biology- Mancuso Page 1 of 4
Lab 6a: Molecular Biology: Electrophoresis ; how gel electrophoresis separates DNA molecules present in a mixture
; the principles of bacterial transformation
; the conditions under which cells can be transformed
; the process of competent cell preparation
; how a plasmid can be engineered to include a piece of foreign DNA
; how plasmid vectors are used to transfer genes
; how antibiotic resistance is transferred between cells
; how restriction endonucleases function
; the importance of restriction enzymes to genetic engineering experiments
Section B: After doing this laboratory you should be able to:
; use plasmids as vectors to transform bacteria with a gene for antibiotic resistance in a controlled
; demonstrate how restrictions enzymes are used in genetic engineering
; use electrophoresis to separate DNA fragments
; describe the biological process of transformation in bacteria
; calculate transformation efficiency
; be able to use multiple experimental controls
; design a procedure to select positively for antibiotic resistant transformed cells ; determine unknown DNA fragment sizes when given DNA fragments of known size
Gel Bed Gel Chamber
Gel Comb EcoRI digest
60 mL 1% agarose gel HindIII digest
Micropipette Phage Lamda DNA
400 mL Buffer Electrical apparatus
Caroline BLU stain Destaining apparatus
Visible light box
PROCEDURE A: CAST AGAROSE GEL
1. Seal ends of gel-casting tray with tape, and insert well-forming comb. Place gel-casting tray out of the way
on lab bench, so that agarose poured in next step can set undisturbed. 2. Carefully pour enough agarose solution into casting tray to fill to a depth of about 6mm. Gel should cover
only about ? the height of the comb teeth. Use the tip of a transfer pipet to move large bubbles or solid
debris to sides or end of tray while gel is still liquid.
3. Gel will become cloudy as it solidifies (about 10 min). Do not move or jar casting tray while agarose is
4. When agarose has solidified, unseal ends of casting tray. Place tray in gel box, so that comb is at negative
5. Fill box with tris-borate-EDTA (TBE) buffer, to level that just covers entire surface of gel. 6. Gently remove comb, taking care not to rip the wells.
7. Make certain that sample wells left by comb are completely submerged. If “dimples” are noticed around
wells, slowly add buffer until they disappear.
8. The gel is now ready to load with DNA. If you will be loading the gel during another period, your teacher
will instruct you to cover the electrophoresis tank to prevent drying of the gel.
PROCEDURE B: LOAD GEL
1. Use transfer pipet to load contents of each supply tube (lambda EcoRI, lambda/HindIII, and lambda only)
into a separate well in gel, as shown in Figure1. Use a fresh pipet for each tube.
AP Biology- Mancuso Page 2 of 4
Lab 6a: Molecular Biology: Electrophoresis
; Steady pipet over well using two hands.
; Be careful to expel any air in pipet tip end before loading gel. (If air bubbles form “cap”
over well, DNA/loading dye will flow into buffer around edges of well.)
; Dip pipet tip through surface of buffer, position it over the wellm and slowly expel the
mixture. Sucrose in the loading dye weighs down the sample, causing it to sink to the
bottom of the well. Be careful not to punch tip of pipet through bottom of gel.
PROCEDURE C: ELECTROPHORESE
1. Close top of electrophoresis chamber, and connect electrical leads to an approved power supply, anode to
anode (red-red) and cathode to cathode (black-black). Make sure both electrodes are connected to same
channel of power supply.
2. Turn on power supply, and set voltage as directed by your teacher. Shortly after current is applied,
loading dye can be seen moving through the gel toward positive pole of electrophoresis apparatus. 3. The loading dye will eventually resolve into two bands of color. The faster moving, purplish band is the
dye bromophenol blue; the slower-moving, aqua band is xylene cyanol.
4. Allow the DNA to electrophorese until the bromophenol blue band nears the end of the gel. Your teacher
may monitor the progress of electrophoresis in your absence.
5. Turn off power supply, disconnect leads from the inputs, and remove top of electrophoresis chamber. 6. Carefully remove casting tray, and slide gel into staining tray labeled with your group name. Take gel to
your instructor for further staining.
HindIII Fragment EcoRI Fragment
Gel A Gel B