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Lab 6 Electrophoresis - Welcome to Guamnet

By Katherine Phillips,2014-05-12 12:01
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Laboratory 6a AP Biology- Mancuso Page 1 of 4 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 a..

     Laboratory

     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).

    OBJECTIVES

    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

    experiment

    ; 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

    MATERIALS

    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

    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

    solidifying.

    4. When agarose has solidified, unseal ends of casting tray. Place tray in gel box, so that comb is at negative

    (black) end.

    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.

    DATA

    HindIII Fragment EcoRI Fragment

    distance distance

    Gel A Gel B Gel A Gel B

    RESULTS AND DISCUSSION

    1. Examine your stained gel on a light box or overhead projector. Compare your gel with the ideal gel

    shown in Figure 1, and try to account for the fragments of lambda DNA in each lane. 2. How can you account for differences in separation and band intensity between your gel and the ideal gel? 3. Two small restriction fragments of nearly the same base-pair size appear as a single band, even when the

    sample is run to the very end of the gel. What could be done to resolve the fragments? Why would it

    work?

    4. Linear DNA fragments migrate at rates inversely proportional to the log10 molecular weights. For

    simplicity’s sake, base-pair length is substituted for molecular weight.

    AP Biology- Mancuso Page 3 of 4

    Lab 6a: Molecular Biology: Electrophoresis

    A) The matrix below gives the actual size in base pairs (Act. Bp) of lambda DNA fragments

    generated by a HindIII digest:

    HindIII EcoRI

     Distance Act. bp Distance Measured bp Act. bp

     *27,491

     *23,130

     9,416

     6,682

     4,361

     2,322

     2,027

     **564

     **125

    *pair appears as single band **does not appear on this gel

    b) Using your gel or the ideal gel shown in Figure 1, carefullt measure the distance (in mm) each

    HindIII and EcoRI fragment migrated from the origin. Measure from the front edge of well to

    front edge of each band. Enter distances into matrix.

    c) Match base-pair sixes of HindIII fragments with bands that appear in the ideal digest. Label each

    band with kilobase-pair (kbp) size. For example, 27,491 bp equals 27.5 kbp.

    d) Set up semilog paper with distance migrated as x (arithmetic) axis and the base-pair length as the

    y (logarithmic) axis. Then, plot distance migrated versusbase-pair length for each HindIII

    fragment.

    e) Connect data points with a best-fit line.

    f) Locate on x axis the distance migrated by the first EcoRI fragment. Using a ruler, draw a vertical

    line from this point to its intersection with a best-fit data line.

    g) Now extend a horizontal line from this point to the y axis. This gives the base-pair size of this

    EcoRI fragment.

    h) Repeat steps f and g for each EcoRI fragment and place answer under Calc. bp after completion,

    your teacher will provide data for Act. Bp for comparison.

    i) For which fragment sizes was your graph most accurate? For which fragment size was it least

    accurate? What does this tell you about the resolving ability of the agarose-gel electrophoresis?

AP Biology- Mancuso Page 4 of 4

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