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EDVO-Kit #
345
Exploring the Genetics of
Taste: SNP Analysis of the
PTC Gene Using PCR
Storage: See Page 3 for
specific storage instructions
EXPERIMENT OBJECTIVE:
The objective of this experiment is for students to
isolate human DNA and use PCR to amplify a segment
of the TAS2R38 gene, which is responsible for detecting
the bitter taste of PTC. Digestion of the PCR products
and analysis by agarose gel electrophoresis are used to
identify the presence of SNP. Genotype is linked to
phenotype by tasting the PTC paper.
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345.130821
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EDVO-Kit #
Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR
345
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Table of Contents
Experiment Components
3
Experiment Requirements
4
Background Information
5
Experiment Procedures
Experiment Overview
11
Module I: Isolation of DNA from Human Cheek Cells
12
Module II: Amplification of the PTC Regions
13
Module III: Restriction Digest of the PTC PCR Products
14
Module IV: Separation of PCR Products and Digestion
Products by Agarose Gel Electrophoresis
Module V: Staining Agarose Gels
15
17
Module VI: Confirmation of Bitter Tasting Ability with PTC Paper
19
Study Questions
20
Instructor’s Guidelines
Pre-Lab Preparations
21
Experiment Results and Analysis
27
Answers to Study Questions
28
Appendices
29
A
Troubleshooting Guides
30
B
Preparation and Handling of PCR Samples With Wax
32
C
Bulk Preparation of Agarose Gels
33
Material Safety Data Sheets can be found on our website:
www.edvotek.com
The PCR process and
Taq DNA polymerase are
covered by patents owned by
Hoffman-LaRoche, Inc.
EDVOTEK, The Biotechnology Education Company, and InstaStain are registered trademarks of
EDVOTEK, Inc. UltraSpec-Agarose, PCR EdvoBead, and FlashBlue are trademarks of EDVOTEK, Inc.
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Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR
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EDVO-Kit #
345
Experiment Components
Component
Storage
A
Room Temperature
❑
-20°C Freezer
-20°C Freezer
-20°C Freezer
-20°C Freezer
Room temperature
-20°C Freezer
-20°C Freezer
-20°C Freezer
Room temperature
Room temperature
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Experiment # 345 contains material
for up to 25 reactions.
Sample volumes are very small.
It is important to quick spin the
tube contents in a microcentrifuge
to obtain sufficient volume for
pipetting. Spin samples for 10-20
seconds at maximum speed.
B
C
D
E
F
G
H
I
•
•
Tubes with PCR EdvoBeads™
Each PCR EdvoBead™ contains
• dNTP Mixture
• Taq DNA Polymerase Buffer
• Taq DNA Polymerase
• MgCl2
• Reaction Buffer
PTC Primer mix concentrate
100 base pair ladder
Control DNA concentrate
TE buffer
Proteinase K
Restriction Enzyme Reaction Buffer
Restriction Enzyme Dilution Buffer
Hae III Restriction Enzyme
PTC Paper
Control Taste Paper
Check (√)
NOTE: Components B and D are supplied in concentrate form and require
dilution prior to setting up PCR reactions.
Reagents & Supplies
Store all components below at room temperature.
Component
All components are intended
for educational research only.
They are not to be used for
diagnostic or drug purposes, nor
administered to or consumed by
humans or animals.
•
•
•
•
•
•
•
•
•
•
•
•
Check (√)
UltraSpec-Agarose™
Electrophoresis Buffer (50x)
10x Gel Loading Solution
InstaStain® Ethidium Bromide
FlashBlue™ Stain
Disposable plastic cups
Conical Tube
Microcentrifuge Tubes (with caps)
Microcentrifuge Tubes (1.5 ml screw-cap tube – use for boiling)
PCR tubes (0.2 ml - for thermal cyclers with 0.2 ml template)
Wax beads (for waterbath option or thermal cyclers
without heated lid)
Salt packets
EDVOTEK - The Biotechnology Education Company®
1-800-EDVOTEK • www.edvotek.com
FAX: 202-370-1501 • email: [email protected]
345.130821
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Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR
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Experiment Requirements
•
*If you do not have a thermal
cycler, PCR experiments can be
conducted, with proper care,
using three waterbaths. However,
a thermal cycler assures a
significantly higher rate of success.
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
(NOT included in this experiment)
Thermal cycler (EDVOTEK Cat. # 541 highly recommended)
or three waterbaths*
Waterbath (55º C and boiling)
(EDVOTEK Cat. # 539 highly recommended)
Horizontal gel electrophoresis apparatus
D.C. power supply
Balance
Microcentrifuge
UV Transilluminator or UV Photodocumentation system (use if staining
with InstaStain® Ethidium Bromide)
UV safety goggles
White light visualization system (optional - use if staining with
FlashBlue™)
Automatic micropipets (5-50 µl) with tips
Microwave or hot plate
Pipet pump
250 ml flasks or beakers
Hot gloves
Disposable laboratory gloves
Ice buckets and ice
Distilled or deionized water
Spring water
Bleach solution
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Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR
EDVO-Kit #
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345
Background Information
SINGLE NUCLEOTIDE POLYMORPHISMS (SNPS)
C
G
C
T
A
G
C
G
1
SNP
2
A
T
T
C
G
C
T
A
A
G
Figure 1: Single Nucleotide
Polymorphism (SNP) occurs
at a single base-pair location
(C/T).
SNPs may fall within any region of the genome – the coding sequences of
genes, the noncoding sequences of genes, or regions in-between genes
(the intergenic regions). When an SNP falls within a coding sequence,
that alteration may not necessarily change the amino acid sequence of
the subsequent protein produced due to degeneracy of the genetic code.
On the other hand, SNPs that do not fall into a coding sequence may still
affect that region of DNA in such a way by inducing gene splicing, transcription factor binding, or by altering the sequence of non-coding RNA.
Variations in our DNA sequences can affect many aspects of human physiology - how we develop diseases, respond to pathogens, process chemicals, drugs , and many more. For example, sickle cell anaemia is caused by
a single nucleotide mutation in the ß-globin chain of hemoglobin. This, in
turn, causes the hydrophilic amino acid glutamic acid to be replaced with
the hydrophobic amino acid valine, thus altering the shape of the blood
cells. Instead of having the usual disc-like shape that is flexible, enabling
them the pass even the smallest blood vessels, sickle cells are long “sickleshaped” red blood cells that block the flow of blood through the blood
vessels. Sickle cells can lead to various complications, including acute
chest syndrome, organ damage, gallstone, and stroke.
THE BITTER TASTE AND THE BITTER COMPOUND
PHENYLTHIOCARBAMIDE (PTC)
Individuals vary greatly in their sensitivity to the bitter compound Phenylthiocarbamide (PTC). This is one of the best-known genetic traits in the human
population and has been a popular teaching tool for genetic inheritance. In this
experiment, we examine variable regions to distinguish differences in individuals
by identifying two different forms (alleles) of a gene related to one’s ability to
taste PTC (Figure 2).
Figure 2:
The structure of PTC.
The famous European geneticist Professor Ed V. Otek, tested his rather large genetics class of students for the ability to taste PTC. He discovered that this gene
has two alleles: the dominant allele (T), which confers the ability to taste PTC,
and the recessive non-taster allele (t). A person inherits one copy of the gene
from each of his / her parents. The combination of these different alleles within
an individual is referred to as a genotype, which in turn dictates the phenotype:
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Background Information
A
T
Single Nucleotide Polymorphisms (SNPs) are DNA sequence variations
that occur in the genome sequence when a single nucleotide is altered.
The genetic code is specified by the four nucleotide letters: A (Adenine),
C (Cytosine), T (Thymine), and G (Guanine). SNP occurs when a single
nucleotide, such as a T, replaces one of the other three nucleotides A, G,
or C (Figure 1). Each person has many SNPs that together create a unique
DNA fingerprint profile for that individual.
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Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR
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Background Information
Background Information
in this case, whether an individual is a “taster” or “non taster.” PTC-tasters have one of two
possible genotypes; either they are homozygous dominant and have two copies of the taster
allele (TT), or they are heterozygous and have one taster allele and one non-taster allele (Tt).
“Non-tasters” are homozygous recessive and have two copies of the non-taster allele (tt).
Within the general population, about 70% of the people tested can taste PTC, whereas the
other 30% cannot.
THE PTC SENSITIVITY GENE – TAS2R38
Studies show that there is an inherited component that effects how people taste PTC. In
2003, more than 70 years after its original discovery, the gene responsible for PTC sensitivity,
TAS2R38, was identified. Analysis of SNPs within the coding region of TAS2R38 revealed that
PTC taster and nontaster alleles differ in 3 amino acids. There are two common forms (or alleles) of the PTC gene: a dominant tasting allele and a recessive non-tasting allele. The shape
of the receptor protein determines how strongly it can bind to PTC. Since all people have
two copies of every gene, combinations of the alleles determine whether someone finds PTC
intensely bitter, somewhat bitter, or tasteless.
As discussed earlier, there are 3 positions within the gene that control the ability to taste PTC
(as shown in table below).
Table I - Relationship
of Variations at Specific
locations in TAS2R38
gene and Ability to
Taste PCR.
Nucleotide
Position
Change in Nucleotide
(Nontaster > Taster)
Change in Codon
(Nontaster > Taster)
Change in Amino Acid
(Nontaster > Taster)
145
G > C
GCA > CCA
Alanine > Proline
785
T > C
GTT
>
GCT
Valine > Alanine
886
A > G
ATC
>
GTC
Valine > Isoleucine
RESTRICTION ENZYME DIGESTION
Restriction enzymes are endonucleases which catalyze the cleavage of the phosphate bonds
within both strands of DNA. The distinguishing feature of restriction enzymes is that they
only cut at very specific sequences of bases. A single base change in the recognition results in
the inability of the restriction enzyme to cut the DNA at that sequence location. Differences
in the sequence of DNA at that specific location can be quickly identified using restriction
enzyme digestion.
A restriction enzyme requires a specific double-stranded recognition sequence of nucleotide
bases to cut DNA. Recognition sites are usually 4 to 8 base pairs in length. Cleavage occurs
within or near the site. Recognition sites are frequently symmetrical, i.e., both DNA strands in
the site have the same base sequence when read 5’ to 3’. Such sequences are called palindromes.
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Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR
EDVO-Kit #
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345
Background Information
Homozygous Taster (TT)
Heterozygous Taster (Tt)
Non-Taster (tt)
Extraction of DNA
from Buccal cells
Extraction of DNA
from Buccal cells
PCR Amplification of
PTC region
PCR Amplification of
PTC region
PCR Amplification of
PTC region
GGCGGCCACT
GGCGGGCACT
GGCGGCCACT
GGCGGGCACT
PCR Product 221 bp
PCR Product 221 bp
PCR Product 221 bp
Hae III Restriction Digest
Hae III Restriction Digest
Hae III Restriction Digest
GGCGG
Figure 3:
Determining PTC
Genotype
CCACT
GGCGGGCACT
GGCGGGCACT
GGCGG
CCACT
Agarose Gel Electrophoresis
Agarose Gel Electrophoresis
44 bp Fragment
177 bp Fragment
Agarose Gel Electrophoresis
221 bp Fragment
(remains uncut)
221 bp Fragment
44 bp Fragment
177 bp Fragment
Lane 1 Lane 2 Lane 3
(TT)
(Tt)
(tt)
221 bp Fragment
177 bp Fragment
PTC TASTER:
Homozygous Taster (TT) = sizes of 177 & 44 bp
Heterozygous Taster (Tt) = 1 allele remains uncut at 221 bp while
the other allele cuts and generates fragments of 177 bp and 44 bp.
PTC NON-TASTER:
Homozygous recessive (tt) = remains uncut at 221 bp
44 bp Fragment
Consider the recognition site and cleavage pattern of EcoRI and HaeIII below. The
cleavage positions are indicated by arrows. Digestion with EcoRI produces assymmetric “sticky ends”, whereas HaeIII restriction enzyme cleavage produces “blunt’ ends.
Cleavage pattern of EcoRI:
↓
5’-GAATTC-3’ Eco RI
3’-CTTAAG-5’ Digestion
↑
5’-G
AATTC-3’
3’-CTTAA
G-5’
Cleavage pattern of HaeIII:
5’-GG CC-3’
3’-CC GG-5’
5’-GG and CC-3’
3’-CC and GG-5’
In the example of the PTC gene, Hae III only cuts the taster allele (5’-GGCGGCCACT-3’). The polymorphism present in the non- taster allele (5’-GGCGGGCACT-3’)
changes a single base change in the restriction enzyme recognition site, so Hae III can
not digest non-taster DNA.
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Background Information
Extraction of DNA
from Buccal cells
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Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR
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Background Information
Background Information
THE POLYMERASE CHAIN REACTION (PCR)
The PCR reaction is a DNA amplification technique that revolutionized almost
all aspects of biological research. The procedure was invented by Dr. Kary Mullis
while at Cetus Corporation in 1984. Dr. Mullis was awarded a Nobel Prize for
his work in 1994. PCR amplification can produce millions of copies from a small
quantity of DNA. The enormous utility of PCR is based on its procedural simplicity
and specificity. Since the first application of PCR to diagnose sickle cell anemia, a
large number of amplifications have successfully been developed. PCR has made
amplification of DNA an effective alternative to cloning. It is currently routinely
used in forensics, paternity/kinship testing, and the identification of human
remains.
In preparation for PCR amplification, a set of two DNA “primers” are designed to
target a specific region of the genomic sequence containing the gene of interest. The primers are two synthetic oligonucleotides typically 15-30 base pairs
in length that are synthesized so that they correspond to the start and end of
a specific region of the DNA sequence to be amplified. In this experiment, the
template DNA is from individuals who show differences in their ability to taste
PTC. The extracted DNA is called the “template.” Freshly isolated DNA from
biological sources will yield the best amplification. DNA extracted from stored
specimens may be degraded and therefore less suitable for amplification.
In addition to the two primers, the four deoxynucleotides (dATP, dCTP, dGTP,
and dTTP) that are the precursors building blocks of DNA and a thermally stable
DNA polymerase are required. The most commonly used DNA polymerase is
the enzyme Taq polymerase, which is purified from the thermophilic bacterium
Thermus aquaticus that inhabits hot springs. This enzyme is stable at near-boiling
temperatures.
The PCR process requires a sequential heating and cooling cycle of the mixture
at three different temperatures. It is efficiently performed in a thermal cycler, an
instrument that is programmed to rapidly heat, cool, and maintain samples at
designated temperatures for varying amounts of time.
In the first step of the PCR reaction the mixture is heated to near boiling
(94°C),to allow the two complementary DNA strands to be denatured. This step,
known as “denaturation,” disrupts the hydrogen bonds between the strands
and causes the complete separation of the two DNA strands. In the second PCR
step, the sample is cooled to a temperature in the range of 45°C - 65°C. In this
step, known as “annealing,” the two primers, which are present in great excess
to the separated DNA template strands, bind to their target complements. In the
third step, known as “extension” (also called DNA synthesis), the temperature
is raised to an intermediate value (usually 72°C). At this temperature the Taq
polymerase is maximally functional. It adds the precursor nucleotides to the primers to complete the synthesis of the new complementary strands based on the
traditional Watson-Crick base pairing. These three steps--denaturation, annealing, and extension constitute one PCR “cycle.” Each cycle doubles the amount of
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Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR
EDVO-Kit #
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345
Background Information
One common problem that occurs during PCR is the production of unwanted
amplification products. This may be due to contamination of the sample or nonspecific annealing (to the wrong segment). If this were to occur in an early cycle,
the incorrect copy will also be amplified. To reduce contamination, autoclaved
tubes and pipet tips, as well as sterile water should be used. Gloves should always
be worn when performing PCR. Minimizing the concentration of the primers may
curtail the production of unwanted PCR due to nonspecific annealing. Another
common technique is a “hot start” step, in which the PCR reagents are introduced in the reaction only after the DNA is fully denatured at 94°C.
In this experiment, the students will use the PCR-RFLP method to examine the
presence of the polymorphism. Students will use the PCR to amplify the polymorphic region of the TAS2R38 gene. The amplified DNA will be digested with
the restriction enzyme Hae III to determine their genotype at position 145,
which correlates with the ability to taste PTC. Agarose gel electrophoresis of the
restriction-digested PCR products will reveal the 2 alleles of the TAS2R38 gene,
indicating whether a student is homozygous or heterozygous for the taster phenotype. In the final module, students will test their ability to taste the bitter PTC
and correlate their genotype with their phenotype.
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The Experiment
the target DNA. Calculated mathematically, if the cycle is repeated n times the
number of copies will be an exponential enlargement of 2n. For example, ten
cycles will produce 210 or 1,048,576 copies. The PCR process is typically repeated
for 20-40 cycles, theoretically amplifying the target sequence to millions of copies. In practice, however, the amount of product often reaches a maximum after
about 35 cycles. This is due to the depletion of reaction components and loss of
Taq polymerase activity. The exact temperature and incubation time required for
each of the three steps depends upon several factors, including the length of the
DNA target and the Guanine /Cytosine (GC) content of the primer/ target.
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Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR
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Background Information
Target Sequence
= Separation of
two DNA strands
5'
3'
3'
5'
5'
3'
3'
5'
Cycle 1
= Primer 2
5'
5'
3'
Cycle 2
3'
3'
3'
5'
Denature
94°C
3'
5'
Anneal
2 primers
40°C - 65°C
5'
5'
3'
5'
5'
5'
3'
Cycle 3
The Experiment
= Primer 1
5'
3'
5'
5'
5'
5'
5'
5'
5'
3'
3'
5'
5'
3'
5'
5'
3'
5'
5'
5'
3'
Extension
72°C
3'
5'
5'
3'
5'
5'
3'
3'
5'
5'
3'
5'
5'
Figure 4 The Polymerase Chain Reaction (PCR) - Three Cycles
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Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR
EDVO-Kit #
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345
Experiment Overview and General Instructions
EXPERIMENT OBJECTIVE
Wear gloves
and safety goggles
MODULE I: 50 min.
IMPORTANT
Isolation of DNA from
Cheek cells
Be sure to READ and UNDERSTAND the instructions completely BEFORE
starting the experiment. If you are unsure of something, ASK YOUR
INSTRUCTOR!
MODULE II: 80 min.
•
Wear gloves and goggles while working in the laboratory.
•
Exercise caution when working in the laboratory – you will be using
equipment that can be dangerous if used incorrectly.
•
Wear protective gloves when working with hot reagents like boiling water and melted agarose.
•
DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS.
•
Always wash hands thoroughly with soap and water after working
in the laboratory.
•
Contaminated laboratory waste (saliva solution, cup, pipet, etc.)
must be disinfected with 15% bleach solution prior to disposal. Be
sure to properly dispose any biological samples according to your
institutional guidelines.
Amplification of the
PTC Regions
MODULE III: 40 min.
Restriction Digest of the
PTC PCR Product
MODULE IV: 50-70 min.
Separation of PCR Products and
Digestion Products by Agarose
Gel Electrophoresis
MODULE V: 5 min.
Staining Agarose Gels
MODULE VI: 10 min.
Confirmation of Bitter Tasting Ability
with PTC Paper
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345.130821
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The Experiment
The objective of this experiment is for students to isolate human DNA
and use PCR to amplify a segment of the TAS2R38 gene, which is
responsible for detecting the bitter taste of PTC. Digestion of the PCR
products and analysis by agarose gel electrophoresis are used to identify
the presence of SNP. Genotype is linked to phenotype by tasting the
PTC paper.
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Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR
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Module I: Isolation of DNA from Human Cheek Cells
1.5 ml
2.
1.
4. SPIN
3.
T.C.
T.C.
60
T.C.
Full speed
2 min.
The Experiment
sec.
5.
Swirl
7.
6.
55° C
Vortex
or Flick
8.
99° C
15
15
140 µl
Lysis
Buffer
min.
min.
99
99
Warning!
Students should
use screw-cap
tubes when boiling
samples.
Vigorously 20 sec.
© 2013 Edvotek® All Rights Reserved.
9.
SPIN
10.
80 µl
Supernatant
Low speed
2 min.
1.
2.
LABEL a 1.5 ml screw top microcentrifuge tube and a cup with your lab group and/or initials.
RINSE your mouth vigorously for 60 seconds using 10 ml saline solution. EXPEL
the solution into cup.
STEP 4:
3. SWIRL the cup gently to resuspend the cells. TRANSFER 1.5 ml of solution into
If cell pellet size is not
large enough, repeat
the labeled tube.
steps 3 - 4 until you
4. CENTRIFUGE the cell suspension for 2 min. at full speed to pellet the cells.
have a large size
POUR off the supernatant, but DO NOT DISTURB THE CELL PELLET! Repeat
pellet. For best results,
steps 3 and 4 twice more.
make sure your cell
pellet is at least the
5. RESUSPEND the cheek cells in 140 µl lysis buffer by pipetting up and down or
size of a match head.
by vortexing vigorously.
6. CAP the tube and PLACE in a waterbath float. INCUBATE the sample in a 55° C
waterbath for 15 min.
STEP 7: If a vortex
is not available, mix
7. MIX the sample by vortexing or flicking the tube vigorously for 20 seconds.
samples by flicking the
8. INCUBATE the sample in a 99° C waterbath for 15 min. Be sure to use screwtube vigorously for 20
cap tubes when boiling DNA isolation samples.
seconds.
9. CENTRIFUGE the cellular lysate for 2 minutes at low speed (6000 rpm).
10. TRANSFER 80 µl of the supernatant to a clean, labeled microcentrifuge tube.
PLACE tube in ice.
11. PROCEED to Module II: Amplification of the TAS2R38 Locus.
OPTIONAL STOPPING POINT:
The extracted DNA may be stored at -20°C for amplification at a later time.
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Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR
EDVO-Kit #
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345
Module II: Amplification of the PTC Regions
2.
1.
• 20 µl PTC primer
• 5 µl extracted DNA
• PCR EdvoBead™
Gently mix
3.
SPIN
#1
4.
5.
If your thermal cycler does not
have a heated lid, it is necessary
to overlay the PCR reaction with
wax to prevent evaporation. See
Appendix B for guidelines.
1. ADD 20 µL PTC primer mix, 5 µl extracted DNA (or Control DNA) and one PCR EdvoBead™ to a
labeled 0.2 ml or 0.5 ml PCR tube (depending on the Thermal Cycler).
2. MIX the PCR sample. Make sure the PCR EdvoBead™ is completely dissolved.
3. CENTRIFUGE to collect the sample at the bottom of the tube.
4. AMPLIFY DNA using PCR:
PCR cycling conditions:
Initial denaturation 94°C for 4 minutes
94° C for 30 seconds
64° C for 45 seconds
35 cycles
72° C for 45 seconds
Final Extension 72° C for 5 minutes
5. PLACE tubes on ice.
PROCEED to Module III: Restriction Digest of PCR Product.
OPTIONAL STOPPING POINT: The PCR samples may be stored at -20° C for restriction
digest at a later time.
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The Experiment
NOTES AND REMINDERS:
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Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR
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Module III: Restriction Digest of the PTC Product
2.
The Experiment
1.
PCR amplified DNA (20µl)
#1
Reaction buffer (5µl)
Hae III Restriction Enzyme (5µl)
4.
SPIN
#1
37° C
30
99
3.
Gently mix.
min.
5.
6.
Gel Loading Dye
(5µl)
#1
Gel Loading Dye (5µl)
Water (15µl)
Digested DNA
#2
Uncut DNA
© 2013 Edvotek® All Rights Reserved.
1. ADD 20 µl PCR amplified DNA, 5 µl Reaction Buffer (F), and 5 µl Restriction Enzyme Hae III to a 1.5 ml
microcentrifuge tube. Label this tube “1”. Save the remaining 5 µl uncut PCR sample to set up as a control
later. Label this as tube “2”.
2. Gently MIX the restriction digest (tube “1”) by gently tapping the tube.
3. Quickly CENTRIFUGE to collect sample at the bottom of the tube.
4. INCUBATE the digest for 30 min at 37°C.
5. ADD 5 µl 10X Gel Loading Dye to 25 µl restriction-digested DNA.
6. ADD 5 µl 10X Gel Loading Dye and 15 µl ultrapure water to 5 µl uncut DNA (tube “2” saved from step 1)
as a control.
PROCEED to Module IV: Agarose Gel Electorphoresis.
OPTIONAL STOPPING POINT: The restriction digests may be stored at -20°C for
electrophoresis at a later time.
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Module IV: Separation of Digestion Products by Agarose Gel Electrophoresis
1.
50
2.
3.
x
Concentrated
buffer
Distilled
water
1:00
IMPORTANT:
In this experiment, students
will perform Agarose Gel
Electrophoresis twice:
Agarose
Flask
4.
5.
• Once for HaeIII Digested
PCR Products (requiring
one 7 x 14 cm gel per
group).
60°C
6.
WAIT
Pour
60°C
1.
2.
3.
4.
5.
6.
7.
• Once for Undigested PCR
Products (requiring one
7 x 7 cm gel per group).
Each gel can be shared
by 4 students. Place wellformer templates (combs)
in the first set of notches.
7.
20
If you are unfamiliar with
agarose gel prep and
electrophoresis, detailed
instructions and helpful
resources are available at
www.edvotek.com
min.
DILUTE concentrated (50X) buffer with distilled water to create 1X buffer (see
Table A).
MIX agarose powder with 1X buffer in a 250 ml flask (see Table B, page 16).
DISSOLVE agarose powder by boiling the solution. MICROWAVE the solution on
high for 1 minute. Carefully REMOVE the flask from the microwave and MIX by
Wear gloves
swirling the flask. Continue to HEAT the solution in 15-second bursts until the
and safety goggles
agarose is completely dissolved (the solution should be clear like water).
COOL agarose to 60° C with careful swirling to promote even dissipation of heat.
While agarose is cooling, SEAL the ends of the gelcasting tray with the rubber end caps. PLACE the well
Table
template (comb) in the appropriate notch.
1x Electrophoresis Buffer (Chamber Buffer)
POUR the cooled agarose solution into the prepared
Dilution
Total Volume
EDVOTEK
Distilled
gel-casting tray. The gel should thoroughly solidify
50x Conc.
Required
Model #
+ Water
Buffer
within 20 minutes. The gel will stiffen and become
6 ml
294 ml
300 ml
M6+
less transparent as it solidifies.
REMOVE end caps and comb. Take particular care
8 ml
392 ml
400 ml
M12
when removing the comb to prevent damage to the
20 ml
980 ml
1000 ml
M36
wells.
A
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The Experiment
Caution! Flask will be HOT!
16
Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR
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345
Module IV: Separation of Digestion Products by Agarose Gel Electrophoresis
8.
9.
Reminder:
Pour
Before loading the samples,
make sure the gel is properly
oriented in the apparatus
chamber.
The Experiment
1X Diluted
Buffer
11.
10.
Wear gloves
and safety goggles
8.
PLACE gel (on the tray) into electrophoresis chamber. COVER the gel with 1X electrophoresis buffer
(See Table B for recommended volumes). The gel should be completely submerged.
9. LOAD the entire sample (30 µL) into the well. RECORD the position of the samples in Table 1, below.
10. PLACE safety cover. CHECK that the gel is properly oriented. Remember, the DNA samples will migrate toward the positive (red) electrode.
11. CONNECT leads to the power source and PERFORM electrophoresis (See Table C for time and voltage
guidelines).
12. After electrophoresis is complete, REMOVE the gel and casting tray from the electrophoresis chamber
and proceed to STAINING the agarose gel.
Table
B
Individual 2.0%
UltraSpec-Agarose™ Gel
Size of Gel
Casting tray
1X Dilluted
Buffer
7 x 7 cm
25 ml
7 x 14 cm
Table
C
50 ml
+
Amt of
Agarose
Table 1
Gel 1: PCR Reaction for PTC Gene
Lane
0.50 g
1.0 g
Time and Voltage Guidelines
(2.0% Agarose Gels)
1
100 bp ladder
2
Undigested PCR Product - Control
3
Undigested PCR Product - Taster 1
4
Undigested PCR Product - Taster 2
5
Undigested PCR Product - Taster 3
6
Undigested PCR Product - Taster 4
Gel 2: RFLP Analysis to Detect PTC Polymorphisms
Time: 7 x 7 cm gel
Time: 7 x 14 cm gel
Volts
~4.0 cm migration
~6.5 cm migration
1
100 bp ladder
125
30 min.
60 min.
2
HaeIII Digested PCR Product - Control
70
60 min.
120 min.
3
HaeIII Digested PCR Product - Taster 1
50
90 min.
150 min.
4
HaeIII Digested PCR Product - Taster 2
5
HaeIII Digested PCR Product - Taster 3
6
HaeIII Digested PCR Product - Taster 4
Lane
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Module V-A: Staining Agarose Gels using InstaStain® Ethidium Bromide
Preferred Method
1.
2.
3.
Moisten
the gel
id
e
.
U.S
5.
d in
Pen
ent
Pat
g
6.
-
InstaStain®
m
Bro
STAIN
300 nm
5
min.
Ethid
nt Pending
U.S. Pate
m Bromide
InstaStain® Ethidiu
U.S. Patent
Pending
WEAR GLOVES AND GOGGLES WHEN USING THIS PRODUCT.
1.
Carefully REMOVE the agarose gel and casting tray from the electrophoresis chamber. SLIDE the gel
off of the casting tray on to a piece of plastic wrap on a flat surface.
DO NOT STAIN GELS IN THE ELECTROPHORESIS APPARATUS.
2.
MOISTEN the gel with a few drops of electrophoresis buffer.
3.
Wearing gloves, REMOVE and DISCARD the clear plastic protective sheet from the unprinted side of
the InstaStain® card(s). PLACE the unprinted side of the InstaStain® Ethidium Bromide card(s) on the
gel. You will need 2 cards to stain a 7 x 14 cm gel.
4.
With a gloved hand, REMOVE air bubbles between the card and the gel by firmly running your fingers
over the entire surface. Otherwise, those regions will not stain.
5.
PLACE the casting tray on top of the gel/card stack. PLACE a small weight (i.e. an empty glass beaker)
on top of the casting tray. This ensures that the InstaStain® Ethidium Bromide card is in direct contact
with the gel surface. STAIN the gel for 3-5 min. for an 0.8% gel or 8-10 min. for a gel 1.0% or greater.
6.
REMOVE the InstaStain® Ethidium Bromide card(s). VISUALIZE the gel using a long wavelength ultraviolet transilluminator (300 nm). DNA should appear as bright orange bands on a dark background.
BE SURE TO WEAR UV-PROTECTIVE EYEWEAR!
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The Experiment
4.
m
idiu
Eth
in®
Sta
sta
In
18
Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR
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Module V-B: Staining Agarose Gels using FlashBlue™
1.
2.
10
x
Concentrated
FlashBlue™ Stain
Wear gloves
and safety goggles
Distilled
water
The Experiment
Flask
3.
5.
4.
STAIN
Pour
5
min.
(-)
1
2
3
4
5
6
DESTAIN
Pour
20
min.
(+)
1.
DILUTE 10 ml of 10x concentrated FlashBlue™ with 90 mL of water in a flask and MIX well.
2.
REMOVE the agarose gel and casting tray from the electrophoresis chamber. SLIDE the gel off of the
casting tray into a small, clean gel-staining tray.
3.
COVER the gel with the 1x FlashBlue™ stain solution. STAIN the gel for 5 minutes. For best results, use
an orbital shaker to gently agitate the gel while staining. STAINING THE GEL FOR LONGER THAN 5
MINUTES WILL REQUIRE EXTRA DESTAINING TIME.
4.
TRANSFER the gel to a second small tray. COVER the gel with water. DESTAIN for at least 20 minutes
with gentle shaking (longer periods will yield better results). Frequent changes of the water will accelerate destaining.
5.
REMOVE the gel from the destaining liquid. VISUALIZE results using a white light visualization system.
DNA will appear as dark blue bands on a light blue background.
Alternate Protocol:
1.
2.
3.
DILUTE one mL of concentrated FlashBlue™ stain with 149 mL dH2O.
COVER the gel with diluted FlashBlue™ stain.
SOAK the gel in the staining liquid for at least three hours. For best results, stain gels overnight.
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Module VI: Confirmation of Bitter Tasting Ability with PTC Paper
For this module, each student should receive the following materials:
•
PTC Paper
•
Control Taste Paper
PROCEDURE:
TASTE the Control strip of paper first.
2.
TASTE the PTC strip of paper.
3.
Compare the taste of the Control and the PTC paper.
•
Notice what the PTC paper tastes like compared to the Control paper: intensly bitter, somewhat bitter, or tasteless.
•
If you are a taster, the PTC paper strip will be bitter. Non-tasters will
not notice a difference between either strip of paper.
ANALYZE THE RESULTS:
1.
Verify the outcome of your bitter tasting ability using the PTC paper
with your genotype in the PCR-RFLP analysis in previous module.
2.
Are you a homozygous bitter taster, a heterozygous bitter taster, or a
nontaster?
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The Experiment
1.
20
EDVO-Kit #
Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR
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Study Questions
The Experiment
Answer the following study questions in your laboratory notebook or on a
separate worksheet.
1.
How is PCR used to determine human genetics and identify polymorphisms in DNA?
2.
What are the three steps in a PCR cycle and what does each step accomplish?
3.
Based on what you have learned about the genotype of TAS2R38 and its
phenotype, fill in the table below:
Genotype
Phenotype
# of DNA bands predicted
TT
Tt
tt
4.
Based on your results, what is your genotype? Why? What is your phenotype? Why? How about your lab partners?
5.
Do the control and PTC paper tasting results correlate with the DNA
digest findings in your ability to taste? How about your lab partner?
6.
Enter your classroom data in a table as shown below:
Phenotype
Genotype
Strong Taster
Weak taster
Nontaster
TT (homozygous)
Tt (heterozygous)
tt (homozygous)
7.
Considering that not everyone who can taste PTC tastes it the same way,
what does this tell you about classical dominant/recessive inheritance?
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Instructor’s Guide
OVERVIEW OF INSTRUCTOR’S PRELAB PREPARATION:
This section outlines the recommended prelab preparations and approximate
time requirement to complete each prelab activity.
ADVANCE PREPARATION:
Preparation For:
Module I:
Isolation of DNA
from Cheek Cells
Module II:
Amplification of
the PTC Regions
Module III:
Restriction Digest of
the PTC PCR Products
What to do:
When:
Time Required:
Prepare and aliquot
various reagents
(Saline, Lysis buffer)
Up to one day before performing the
experiment. IMPORTANT: Prepare the Lysis
buffer no more than one hour before
performing the experiment.
30 min.
Equilibrate waterbaths
at 55° C and boiling.
One hour before performing
the experiment.
5 min.
Prepare and aliquot various
reagents (Primer, DNA
template, ladder, etc.)
One day to 30 min. before performing
the experiment.
30 min.
Program Thermal Cycler
One hour before performing
the experiment.
15 min.
Prepare and aliquot various
reagents (Reaction buffer,
HaeIII Restriction enzyme, etc.)
Equilibrate waterbath at 37° C
One day to 30 min. before performing
the experiment. IMPORTANT: Prepare the
diluted HaeIII Restriction Enzyme no more than
one hour before performing the experiment.
30 min.
One hour before performing
the experiment.
15 min.
Up to one day before performing
the experiment.
45 min.
Module IV: Separation
of PCR Products &
Digestion Products by
Electrophoresis
Prepare diluted TAE buffer
Module V:
Staining Agarose Gels
Prepare staining
components
The class period or overnight after the
class period.
10 min.
Module VI:
Confirmation of
Bitter Tasting Ability
Distribute PTC taste strips and
Control taste strips
The class period
5 min.
Prepare molten agarose
and pour gel
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Pre-Lab Preparations: Module I
Instructor’s Guide
ISOLATION OF DNA FROM HUMAN CHEEK CELLS
FOR MODULE I
Each Student should receive:
• One cup containing 10 ml of
saline solution
• One screw-cap tube
• One microcentrifuge tube
Reagents to be Shared by
Two Students:
• 300 µl Lysis buffer
• 15% bleach solution
Preparation of Saline Solution
1.
To prepare the saline solution, dissolve all 8 salt packets in 500 ml of
drinking water. Cap and invert bottle to mix.
2.
Aliquot 10 ml of saline solution per cup. Distribute one cup per student.
Preparation of Lysis Buffer
(Prepared no more than one hour before starting the experiment.)
1.
Add 100 µl of TE buffer (E) to the tube of Proteinase K (F) and allow the
sample to hydrate for several minutes. After the sample is hydrated,
pipet up and down several times to thoroughly mix the material.
2.
Transfer the entire amount of the rehydrated Proteinase K solution to a
15 ml conical tube containing an additional 4 ml of TE buffer (E).
3.
Invert the tube several times to mix. Label this tube “Lysis Buffer”.
4.
Aliquot 300 µl of Lysis Buffer into 13 labeled microcentrifuge tubes.
5.
Distribute one tube of “Lysis Buffer” to each student pair.
Warning !!
Remind students to only use
screw-cap tubes when boiling
their DNA samples. The snap-top
tubes can potentially pop open
and cause injury.
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Pre-Lab Preparations: Module II
Preparation of the PTC Primer
Reagents to be Shared by
Two Students:
• 50 µl PTC Primer mix
1.
Thaw the PTC Primer Mix Concentrate (B) on ice.
2.
Add 1 ml of TE Buffer (E) to the tube of Primer Mix Concentrate. Cap
tube and mix.
3.
Label 13 microcentrifuge tubes “PTC Primer”. Aliquot 50 µl of the
diluted Primer Mix into the 13 microcentrifuge tubes. Place the tubes
on ice until they are needed.
4.
Distribute one tube of diluted PTC Primer to each student pair.
Preparation of the Control DNA
1.
This kit includes enough DNA to set up 4 control reactions. At least
one control reaction should be performed per class to confirm that
PCR was successful.
2.
Thaw the tube of Control DNA Concentrate (D) on ice.
3.
Add 20 µl of TE buffer (E) to the tube containing Control DNA Concentrate. Pipet up and down to mix.
4.
Dispense 8 µl of the diluted control DNA for each control reaction.
Preparation of Additional Materials
1.
Dispense 30 µl of 10x Gel Loading Solution for each student pair.
Programming the Thermal Cycler
The Thermal cycler should be programmed as outlined in Module II in the
Student’s Experimental Procedure.
•
Accurate temperatures and cycle times are critical. A pre-run for one
cycle (takes approximately 3 to 5 min) is recommended to check that
the thermal cycler is properly programmed.
•
For thermal cyclers that do not have a heated lid, it is necessary to
place a layer of wax above the PCR reactions in the microcentrifuge
tubes to prevent evaporation. See Appendix B for instructions.
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Instructor’s Guide
FOR MODULE II
Each Student should receive:
• One PCR tube and PCR
EdvoBead™
• 30 µl Gel Loading Solution
24
EDVO-Kit #
Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR
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Pre-Lab Preparations: Module III
Instructor’s Guide
MODULE III: RESTRICTION DIGEST OF THE PTC PRODUCT
FOR MODULE III
Reagents to be Shared by
Two Students:
• 12 µl HaeIII Restriction Enzyme
• 15 µl Restriction Enzyme
Reaction Buffer
• 20 µl 10X Gel Loadion Solution
Dilution of HaeIII restriction enzyme
1.
Add 150 µl of Restriction Enzyme Dilution Buffer (H) to the tube containing the concentrated HaeIII restriction enzyme.
2.
Mix the tube for 30 seconds (vortex or tap bottom of the tube) and set
on ice 1 minute.
3.
Dispense 12 µl of the HaeIII Restriction Enzyme for 13 tubes. Label these
tubes “HaeIII”
4.
Place the tubes on ice until they are needed. Each tube will be shared by
a student pair.
Also aliquot the following reagents:
1.
Dispense 15 µl of Restriction Enzyme Reaction Buffer (G) per student
pair.
2.
Dispense 20 µl of 10X Gel Loading Solution per student pair.
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Pre-Lab Preparations: Module IV
If students are unfamiliar with
using micropipets, we recommended performing Cat. #S-44,
Micropipetting Basics or Cat.
#S-43, DNA DuraGel™ prior to
conducting this advanced level
experiment.
FOR MODULE IV
Each Student Group
should receive:
• 50x concentrated buffer
• Distilled Water
• UltraSpec-Agarose™
• Tube of 100 bp ladder (65 µl
per 2 gels shared by 4-5
students per gel.)
SEPARATION OF PCR PRODUCTS AND DIGESTION PRODUCTS
BY AGAROSE GEL ELECTROPHORESIS
This experiment requires two 2.0% agarose gel per student group. Each
group of 4-5 students will share one 7 x 7 cm and one 7 x 14 cm gel.
•
A 7 x 7 cm gel is recommended for separating undigested PCR Products.
•
A 7 x 10 cm or 7 x 14 cm gel is recommended for separating Hae III digest of PCR Products.
You can choose whether to prepare the gels in advance or have the students
prepare their own. Allow approximately 30-40 minutes for this procedure.
Individual Gel Preparation:
Each student group can be responsible for casting their own individual gel
prior to conducting the experiment. See Module III in the Student’s Experimental Procedure. Students will need 50x concentrated buffer, distilled
water and agarose powder.
Batch Gel Preparation:
To save time, a larger quantity of agarose solution can be prepared for sharing by the class. See Appendix C.
Preparing Gels in Advance:
Gels may be prepared ahead and stored for later use. Solidified gels can be
store under buffer in the refrigerator for up to 2 weeks.
Do not freeze gels at -20ºC as freezing will destroy the gels.
Gels that have been removed from their trays for storage should be “anchored” back to the tray with a few drops of molten agarose before being
placed into the tray. This will prevent the gels from sliding around in the
trays and the chambers.
Additional Materials:
•
Each set of two 2.0% gel should be loaded with the 100 base pair ladder
and samples from 4 or 5 students. The control PCR reaction can also be
loaded in one of the wells.
•
Aliquot 65 µl of the 100 base-pair ladder (C) into labeled microcentrifuge tubes.
•
Distribute one tube of ladder per two 2.0% agarose gels shared by 4-5
students as outlined above.
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Instructor’s Guide
NOTE:
Accurate pipetting is critical for
maximizing successful experiment results. EDVOTEK Series 300
experiments are designed for
students who have had previous
experience with micropipetting
techniques and agarose gel
electrophoresis.
26
EDVO-Kit #
Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR
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Instructor’s Guide
Pre-Lab Preparations: Module V
FOR MODULE IV
Each Student Group
should receive:
• 2 InstaStain® cards per
7 x 14 cm gel
Wear gloves
and safety goggles
STAINING WITH INSTASTAIN® ETHIDIUM
BROMIDE
Preferred Method
InstaStain® Ethidium Bromide provides the sensitivity of ethidium bromide
while minimizing the volume of liquid waste generated by staining and
destaining a gel. An agarose gel stained with InstaStain® Ethidium Bromide
is ready for visualization in as little as 3 minutes! Each InstaStain® card will
stain 49 cm2 of gel (7 x 7 cm). You will need 2 cards to stain a 7 x 14 cm gel.
Use a mid-range ultraviolet transilluminator (Cat. #558) to visualize gels stained with
InstaStain® Ethidium Bromide. BE SURE TO WEAR UV-PROTECTIVE EYEWEAR!
•
Standard DNA markers should be visible after staining even if other DNA samples
are faint or absent. If bands appear faint, repeat staining with a fresh InstaStain
card for an additional 3-5 min. If markers are not visible, troubleshoot for problems
with electrophoretic separation.
•
Ethidium bromide is a listed mutagen. Wear gloves and protective eyewear when
using this product. UV protective eyewear is required for visualization with a UV
transilluminator.
•
InstaStain® Ethidium Bromide cards and stained gels should be discarded using
institutional guidelines for solid chemical waste.
STAINING WITH FLASHBLUE™
FlashBlue™ can be used as an alternative to Ethidium Bromide in this experiment.
However, FlashBlue™ is less sensitive than InstaStain® Ethidium Bromide and will take a
longer time to obtain results.
FlashBlue™ stain, however, is optimized to shorten the time required for both staining
and destaining steps. Agarose gels can be stained with diluted FlashBlue™ for 5 minutes
and destained for only 20 minutes. For the best results, leave the gel in liquid overnight.
This will allow the stained gel to “equilibrate” in the destaining solution, resulting in
dark blue DNA bands contrasting against a uniformly light blue background. A white
light box (Cat. #552) is recommended for visualizing gels stained with FlashBlue™.
•
Stained gels may be stored in destaining liquid for several weeks with refrigeration,
although the bands may fade with time. If this happens, re-stain the gel.
•
Destained gels can be discarded in solid waste disposal. Destaining solutions can be
disposed of down the drain.
PHOTODOCUMENTATION OF DNA (OPTIONAL)
Once gels are stained, you may wish to photograph your results. There are many different photodocumentation systems available, including digital systems that are interfaced
directly with computers. Specific instructions will vary depending upon the type of photodocumentation system you are using.
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Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR
EDVO-Kit #
27
345
Experiment Results and Analysis
Gel 1 – PCR Reaction for PTC gene
PCR Products resulted from amplification of the PTC regions showed
that, without the use of restriction enzymes, DNA from different individuals containing an identical region of a chromosome appear to
be similar because no differences in the restriction fragment lengths
are observed.
Gel 2: RFLP Analysis to Detect PTC Polymorphisms
Lane
1 100 bp ladder
2 HaeIII digest of PCR Product - Control
3 HaeIII digest of PCR Product - Taster 1 (example)
4 HaeIII digest of PCR Product - Taster 2 (example)
5 HaeIII digest of PCR Product - Taster 3 (example)
6 HaeIII digest of PCR Product - Taster 4 (example)
•
HaeIII digest of PCR Product Taster 1 (example) shows that Taster
1 is a TT Homozygous Taster. Since HaeIII restriction enzyme
digests the sequence, there are two bands (a bright one at 177
bp and a fainter one at 44 bp)
•
HaeIII digest of PCR Product Taster 2 (example) shows that this
taster is a Tt Heterozygous Taster: 3 bands (3 fragments at 221
bp, 177 bp and 44 bp) This is because half of the taster’s DNA
has the taster allele, and the other half is made up of the nontaster allele.
•
HaeIII digests of PCR Product Tasters 3 & 4 (examples) show that
they are non-tasters. Because HaeIII restriction enzyme cannot
digest the non-taster sequence, PCR product will remain intact
and results in one band at 221 bp.
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Instructor’s Guide
Lane
1 100 bp ladder
2 Undigested PCR Product - Control
3 Undigested PCR Product - Taster 1
4 Undigested PCR Product - Taster 2
5 Undigested PCR Product - Taster 3
6 Undigested PCR Product - Taster 4
Please refer to the kit
insert for the Answers to
Study Questions
Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR
EDVO-Kit #
345
Appendices
A
EDVOTEK® Troubleshooting Guide
B
Preparation and Handling of PCR Samples With Wax
C
Bulk Preparation of Agarose Gels
Material Safety Data Sheets:
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FAX: 202-370-1501 • email: [email protected]
345.130821
29
30
Appendix A
EDVO-Kit #
345
EDVOTEK® Troubleshooting Guides
DNA EXTRACTION
PROBLEM:
There is no cell pellet
after centrifuging the
cheek cell suspension.
Poor DNA Extraction
The extracted DNA is
very cloudy.
CAUSE:
ANSWER:
Not enough cheek cells in suspension
Mouth must be vigorously rinsed for at least 60 sec.
to harvest loose cheek cells.
Sample not centrifuged fast enough
Spin cells at maximum speed (17,000 x g) for 2 min.
If your centrifuge does not reach this speed, spin at
highest available speed for 4 min.
Samples not mixed well enough during
extraction
In addition to flicking the tube, vortex or pipet up and
down to mix the sample.
Proteinase K inactive because it was
prepared too far in advance.
Prepare Proteinase K within one hour of use.
Water baths not at proper temperature
Use a thermometer to confirm water bath set point.
Not enough DNA
Try cheek cell extraction. Final DNA concentrations
are usually higher.
Cellular debris from pellet transferred to
tube
Centrifuge sample again and move supernatant to a
fresh tube. Take care to avoid pellet.
Cellular debris not separated from
supernatant
Centrifuge sample again. If possible, centrifuge at a
higher speed. Move cleared supernatant to a fresh
tube.
RESTRICTION ENZYME DIGESTION
PROBLEM:
CAUSE:
ANSWER:
Impure DNA – some contaminants (EDTA,
salts) might partially or completely inhibit
activity of Hae III restriction enzyme
Poor DNA extraction. Extract new DNA. Cheek cell
extraction usually results in higher DNA yield.
Improper dilution of enzyme
Ensure that Hae III restriction enzyme was correctly
diluted.
Improper addition of enzyme
Ensure that correct amount of Hae III restriction
enzyme was added to the restriction digest.
Incorrect incubation temperature
Use a thermometer to confirm water bath
temperature and adjust, if necessary.
Unexpected Cleavage
Pattern
DNA sample is contaminated
Prepare a new DNA sample.
Smearing of digested
DNA on gel
Nuclease contamination
Care should be taken to avoid cross contamination
when setting up reactions.
Undigested or
incompletely digested
DNA
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345.130821
Use fresh electrophoresis buffer and appropriate
all rights reserved.
Agarose running
conditions
voltage.
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345.130821
Appendix A
31
EDVO-Kit #
345
EDVOTEK® Troubleshooting Guides
PCR AND ELECTROPHORESIS
PROBLEM:
CAUSE:
ANSWER:
Make sure the heated lid reaches the appropriate temperature.
There is very little liquid
left in tube after PCR
Sample has evaporated
If your thermal cycler does not have a heated lid, overlay the
PCR reaction with wax (see Appendix B for details)
Make sure students close the lid of the PCR tube properly.
Pipetting error
Make sure students pipet 20 µL primer mix and 5 µL extracted
DNA into the 0.2 mL tube.
Ensure that the electrophoresis buffer was correctly diluted.
The gel was not prepared properly.
Gels of higher concentration (> 0.8%) require special attention
when melting the agarose. Make sure that the solution is
completely clear of “clumps” and glassy granules before
pouring gels.
The gel was not stained properly.
Repeat staining.
Malfunctioning electrophoresis unit or
power source.
Contact the manufacturer of the electrophoresis unit
or power source.
The gel was not stained for a sufficient
period of time.
Repeat staining protocol.
The ladder, control DNA,
and student PCR products
are not visible on the gel.
After staining the gel,
the DNA bands are faint.
After staining, the ladder
is visible but no PCR
products are present.
Repeat PCR with fresh PCR EdvoBeads™ and primers.
PCR amplification was unsuccessful.
Ensure that the thermal cycler has been properly
programmed. See Module II for guidelines
Student DNA sample was not concentrated
enough.
Poor DNA extraction. Extract new DNA. Cheek cell
extraction usually results in higher DNA yield.
Student DNA sample was degraded
If DNA is not used immediately following extraction, store
sample at -20°C.
Wrong volumes of DNA and primer added
to PCR reaction
Practice using pipettes
Some students have more
or less amplification than
others.
Concentration of DNA varies by sample.
There is an inherent variability in the extraction
process. For best results, use cheek cell extraction.
Low molecular weight
band in PCR samples
Primer dimer
Low concentration of extracted DNA in PCR reaction.
DNA bands were not
resolved.
Tracking dye should migrate at least 3.5 cm
(if using a 7x7 cm tray), and at least 6 cm
(if using a 7x14 cm tray) from the wells to
ensure adequate separation.
Be sure to run the gel at least 6 cm before staining
and visualizing the DNA (approximately one hour at
125 V).
DNA bands fade when
gels are kept at 4°C.
DNA stained with FlashBlue™ may
fade with time
Re-stain the gel with FlashBlue™
After staining, the ladder
and control PCR products
are visible on gel, but
some student samples
are not present.
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all rights reserved.
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345.130821
32
EDVO-Kit #
Appendix B
345
Preparation and Handling of PCR Samples With Wax
ONLY For Thermal Cyclers WITHOUT Heated Lids, or Manual PCR Using Three Waterbaths
Using a wax overlay on reaction components prevents evaporation during the PCR process.
HOW TO PREPARE A WAX OVERLAY
1.
Add PCR components to the 0.2 ml PCR Tube as outlined in Module III.
2.
Centrifuge at full speed for five seconds to collect sample at bottom of the tube.
3.
Using clean forceps, add one wax bead to the PCR tube.
4.
Place samples in PCR machine and proceed with Module III.
PREPARING PCR SAMPLES FOR ELECTROPHORESIS
1.
After PCR is completed, melt the wax overlay by heating the sample at 94° C for three
minutes or until the wax melts.
2.
Using a clean pipet, remove as much overlay wax as possible.
3.
Allow the remaining wax to solidify.
4.
Use a pipet tip to puncture the thin layer of remaining wax. Using a fresh pipet tip,
remove the PCR product and transfer to a new tube.
5.
Add 5 µL of 10x Gel Loading Buffer to the sample. Proceed to Module IV to perform
electrophoresis.
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345.130821
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33
Appendix C
EDVO-Kit #
345
Bulk Preparation of Agarose Gels
To save time, electrophoresis buffer and agarose gel solution can be prepared in larger quantities for sharing
by the class. Unused diluted buffer can be used at a later time and solidified agarose gel can be remelted.
BULK ELECTROPHORESIS BUFFER
Bulk preparation of 1X electrophoresis buffer is
outlined in Table D.
BATCH AGAROSE GELS (2.0%)
Table
D
Bulk Preparation of Electrophoresis Buffer
50x Conc.
Buffer
+
60 ml
Distilled
Water
Total Volume
Required
2,940 ml
3000 ml (3 L)
Bulk preparation of 2.0% agarose gel is outlined in Table E.
Note:
The UltraSpec-Agarose™ kit
component is usually labeled
with the amount it contains.
Please read the label carefully.
If the amount of agarose is not
specified or if the bottle's plastic
seal has been broken, weigh the
agarose to ensure you are using
the correct amount.
1.
Use a 500 ml flask to prepare the diluted gel buffer.
2.
Pour the appropriate amount of UltraSpec-Agarose™ into the prepared buffer.
Swirl to disperse clumps.
3.
With a marking pen, indicate the level of solution volume on the outside of the
flask.
4.
Heat the agarose solution as outlined previously for individual gel preparation.
The heating time will require adjustment due to the larger total volume of gel
buffer solution.
5.
Cool the agarose solution to 60°C with swirling to promote even dissipation of heat. If evaporation
has occurred, add distilled water to bring the solution up to the original volume as marked on the
flask in step 3.
6.
Dispense the required volume of cooled agarose solution for casting each gel. The volume required
is dependent upon the size of the gel bed.
7.
Allow the gel to completely solidify. It will become firm and cool to the touch after approximately
20 minutes. Proceed with electrophoresis (Module IV) or store the gels at 4ºC under buffer.
60˚C
Table
E
Batch Prep of 2.0%
UltraSpec-Agarose™
Amt of
Agarose
8.0 g
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+
Diluted
Buffer (1x)
400 ml