Download 18 DNA and Biotechnology

Laboratory
18
DNA and Biotechnology
(LM pages 243–254)
Time Estimate for Entire Lab: 2.5–3.0 hours
Special Requirements
1. Incubation for Experimental Procedure: Bacterial Transformation (LM page 253). Subculture
E. coli onto Luria broth agar at least 48 hours prior to lab.
2. Overnight preparation time. Onion filtrate for Experimental Procedure: Isolating DNA (LM
page 250).
3. Fume hood. Use for Experimental Procedure: Isolating DNA (LM page 250).
4. Demonstration (instructor-conducted) of Experimental Procedure: Bacterial Transformation (LM
page 253).
Seventh Edition Changes
This was lab 17 Molecular Genetics in the previous edition. The title was changed to correspond to the text.
MATERIALS AND PREPARATIONS1
18.1 DNA Structure and Replication (LM pages 244-245)
_____
model, DNA; model kit, DNA-RNA; or puzzle kit, DNA (Carolina 17-1050)
Models. A wide range of DNA model kits is available from Carolina Biological Supply. The kits vary in
degree of sophistication and in price. Descriptions and price information can be found in the “Genetics” section of the Carolina catalog.
18.3 Isolation and Testing for DNA (LM page 250)
_____
test tubes, standard size, three
_____
test-tube racks
_____
wax pencils
_____
95% ethanol (Carolina 86-1281), ice cold (5 ml per student group)
_____
4% salt (NaCl) (Carolina 88-8880, -8901)
_____
water, distilled
_____
graduated cylinder, glass, 10 ml
_____
onion filtrate:
_____ onion, fresh
_____ salt (NaCl)
_____ dishwashing detergent
_____ water bath, ice
_____ blender
_____ spoon
_____ cheesecloth or #6 coffee filter
_____ beakers, 500 ml and 1,000 ml
_____ large funnel
_____
water baths, hot and boiling:
_____ hot plate
_____ large beakers
_____ beaker tongs
_____ thermometer, Celsius (0–110°C)
1 Note: “Materials and Preparations” instructions are grouped by exercise. Some materials may be used in more than one exercise.
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_____
_____
_____
_____
test tube, large
Pasteur pipette
chloroform (Carolina 85-3980, -4000)
diphenylamine solution:
_____ diphenylamine crystals (Carolina 85-8328, -8330)
_____ acetic acid, glacial (Carolina 84-1292)
_____ sulfuric acid, concentrated (Carolina 89-3391)
_____ water, distilled
_____ acetaldehyde (Carolina 84-1200)
_____ dark storage bottle
4% salt (NaCl) solution. Add 4 g of NaCl to 100 ml of distilled water.
Onion filtrate. Cut one large onion into medium-large pieces, and place the pieces in a 500 ml beaker. Cover
the onion with 100 ml of a NaCl–detergent solution. (To prepare the NaCl–detergent solution, add 15 g of
NaCl to 100 ml of dishwashing detergent (any grocery brand will do), and dilute to 1,000 ml.) Stir mixture
and let stand 15 minutes in a hot water bath at 60° C. (Temperature is not critical, but do not have the water
boiling. Your water bath can be a sink containing hot tap water.) Cool the mixture in an ice-water bath for 5
minutes, stirring frequently with a spoon. Pour the mixture into a blender, and blend for 1 minute on low
speed, then 30 seconds on high speed. Place four thicknesses of cheesecloth (or a #6 coffee filter) in a large
funnel or colander over a large beaker. (Be careful, and try to prevent foam from getting into the filtrate.)
Put the whole setup into the refrigerator, and let it filter overnight.
Chloroform. 5 ml per student group is sufficient. Store in an explosion-proof refrigerator.
Diphenylamine solution. 10 ml per student group is sufficient. Prepare a stock solution by dissolving 16 g
of diphenylamine crystals in 1,000 ml of glacial acetic acid. Add 15 ml of concentrated sulfuric acid. Store in
a dark bottle. On day of use, prepare a fresh solution of acetaldehyde by adding 1 ml of acetaldehyde to 500
ml of distilled water. Prepare the working diphenylamine solution by adding 1 ml of fresh acetaldehyde
solution to every 100 ml of diphenylamine solution.
18.4 Bacterial Transformation (LM pages 251-253)
_____
Luria broth agar plates, prepoured, ready-to-use (Carolina 21-6600) or Luria broth agar,
premixed, ready-to-pour (Carolina 21-6620) and sterile petri plates
_____
Luria broth with ampicillin (Carolina 21-6621), sterile
_____
15% chlorine (bleach) solution, or 10% Lysol solution, or an autoclave
_____
transformation tubes (Carolina 21-5090), sterile, capped, three per student group
_____
test tube racks
_____
wax pencils
_____
dropping pipettes, plastic, 4 ml sterile, calibrated (Carolina 73-6990), four per student
group
_____
styrofoam coffee cups, three per student group
_____
ice, crushed
_____
50 mM calcium chloride (CaCl2) solution (Carolina 21-7260)
_____
water, distilled
_____
Bunsen burner and inoculating loop
_____
Escherichia coli, strain MM294 (Carolina 21-1530)
_____
plasmid DNA (pAMP) (Carolina 21-1429)
_____
water bath, hot/cold tap
_____
thermometer, Celsius (0–110°C)
_____
stopwatch
_____
gloves, plastic or latex
Bacterial transformation. You will need capped transformation tubes (these tubes are specified as they
allow rapid heat transfer during the heat shock phase). Also needed are calibrated, sterile droppers; Luria
broth agar plates, Luria broth with ampicillin, rapidly growing Escherichia coli strain, and plasmid DNA
(pAMP).
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1. Using sterile technique, subculture the E. coli strain by streaking it onto the surface of a Luria agar
plate. Incubate plates for 48 hours at 37°C. Several plates can be inoculated from the same slant tube culture.
Allow one to two plates per lab class.
2. Any lab materials that have contacted E. coli should be placed in a 15% chlorine bleach solution or
a 10% Lysol solution overnight. Pressure-cooking the materials at 15 PSI for 15 minutes or autoclaving are
alternative methods of sterilization.
3. Practice filling the sterile droppers to various levels before the laboratory day. Use one of these droppers to dispense the one drop of plasmid DNA.
4. You do not have to do bacterial transformation to demonstrate the experiment: Number three test
tubes. Add water to all three and starch to the first so that it looks cloudy. Set the tubes aside, where students
cannot see them. Walk the students through the experiment, preferably having on the front bench all of the
necessary laboratory hardware and tubes, marked as if they contain the necessary solutions. In particular,
demonstrate sterile technique (there is no need to have E. coli). Have the students complete Table 18.6. Then
bring out the set-aside tubes, as if 48 hours have passed. Ask the students what conclusions they would
draw if any tube was cloudy.
Calcium chloride (CaCl2) solution. To prepare this solution, add 2.75 g of CaCl2 for every 500 ml of distilled water. Autoclave this solution at 15 pounds for 15 minutes, and refrigerate it until ready for use. This
solution keeps indefinitely.
Escherichia coli. E. coli is shipped in a slant tube culture that can be refrigerated for six months. Subculture
E. coli onto Luria broth at least 48 hours prior to the lab.
Plasmid DNA (pAMP). Plasmid DNA is shipped in concentrated, aqueous form. The plasma comes with
TE buffer and should be diluted in a ratio of one drop plasmid to nineteen drops buffer. Diluted DNA
remains viable for long periods if refrigerated.
EXERCISE QUESTIONS
18.1 DNA Structure and Replication (LM pages 244-245)
Observation: DNA Structure (LM page 244)
2. With reference to DNA’s ladder-type structure, what molecules make up the sides of the ladder?
sugar and phosphate molecules What molecules make up the ladder rungs? hydrogen-bonded bases
adenine, cytosine, guanine, and thymine
3. What is this called (when A pairs with T, and G pairs with C)? complementary base pairing
Observation: DNA Replication (LM page 245)
Explain why each double helix is exactly the same as the other one. Each new DNA is composed of an old
parental strand and a complementary new daughter strand.
18.2 DNA and Protein Synthesis (LM pages 246-249)
Observation: Transcription (LM page 246)
Table 18.1 Transcription
DNA
TAC
GGG
CTA
CAA
CTT
AAC
AGA
CCA
ATC
mRNA
AUG
CCC
GAU
GUU
GAA
UUG
UCU
GGU
UAG
2. Why are the bases in Table 18.1 shown in groups of three? The genetic code is a triplet code.
3. Every three bases in mRNA is called a codon.
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Translation (LM page 248)
Observation: Translation (LM page 248)
3. State the order of the amino acids in the polypeptide you created. proline—aspartate—valine—
glutamate—leucine—serine
Table 18.2 Translation
DNA
TAC
GGG
CTA
CAA
mRNA
tRNA
Amino
acid
CTT
AAC
AGA
ATC
AUG
CCC
GAU
UAC
GGG
CUA
GUU
GAA
UUG
UCU
UAG
CAA
CUU
AAC
AGA
AUC
methionine
(START)
proline
aspartate
valine
glutamate
leucine
serine
STOP
18.3 Isolation and Testing for DNA (LM page 250)
Experimental Procedure: Isolating DNA (LM page 250)
Table 18.4 Testing for DNA
Tube
Tube Contents
Color Change
Explanation
2
DNA
NaCl
Diphenylamine
Blue-green
DNA is present.
3
Water
Diphenylamine
Light yellow
DNA is not present.
This is the control tube.
18.4 Bacterial Transformation (LM pages 251-253)
Questions (LM page 252)
1. What will be considered positive results in the experiment shown in Figure 18.6? growth of E. coli
2. Based on your study of Figure 18.5, which tube in this experiment will show positive results? the
tube marked [] Explain. Plasmid contains resistant gene.
3. Why don’t you expect the tube marked [–] to give positive results? no plasmid
4. Why is the tube marked [C] considered the control? no E. coli
Experimental Procedure: Bacterial Transformation (LM page 253)
Table 18.5 Bacterial Transformation
Tube
E. coli
pAMP
+
Yes
Yes
_
Yes
No
C
No
Yes
Yes
*Luria broth with ampicillin
LB + AMP*
Expected
Results
Conclusions
Yes
Growth
E. coli was transformed.
Yes
No growth
E. coli was not transformed because this tube
contains no plasmid.
No growth
This tube contains no E. coli.
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LABORATORY REVIEW 18 (LM page 254)
1. The DNA structure resembles a twisted ladder. What molecules make up the sides of the ladder?
sugar and phosphate
2. What makes up the rungs of the ladder? hydrogen-bonded bases
3. Do the two DNA double helices following DNA replication have the same, or a different, composition? same
4. What base is adenine paired with? thymine
5. What three types of RNA are involved in protein synthesis? mRNA, rRNA, tRNA
6. If the codons are AUG, CGC, and UAC, what are the anticodons? UAC, GCG, AUG
7. During transcription, DNA is a template for RNA formation.
8. When DNA is present, diphenylamine changes to what color? green
9. In what part of the cell does translation occur? in the cytoplasm
10. What must bacteria take up before they can be transformed? plasmid
11. What term is used to describe recombined DNA from two different sources? recombinant DNA
12. What is a self-replicating circle of extrachromosomal DNA? plasmid
Thought Questions
13. What are the roles of mRNA in transcription and translation? In transcription, mRNA is formed when
nucleotides complementary to the sequences of bases in a portion of DNA join. In translation, the codons of
mRNA code for the sequence of amino acids in a protein.
14. In this laboratory, the plasmid designated as pAMP was used in transformation of E. coli. What
was the test to show that transformation (uptake of the plasmid) had occurred? Bacterial growth on
agar plates containing ampicillin indicates that a recombinant plasmid was taken up by an E. coli cell.