Download Polymerase Chain Reaction and PTC lab

Polymerase Chain Reaction and PTC lab
AP Biology
Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR)
The polymerase chain reaction, PCR, can produce many copies of a specific target
segment of DNA
A three-step cycle—heating, cooling, and replication—brings about a chain reaction
that produces an exponentially growing population of identical DNA molecules
PCR reagents
Primer: 15-20 bp segment complementary to ends of the DNA strand
Taq polymerase: DNA polymerase from Thermus aquaticus bacteria (live in hot
springs)
Supply of free nucleotide triphosphates
PCR steps
1. Denature DNA by heating to 95oC to separate strands
2. Anneal primers by cooling to 37oC so primers bond to DNA strands
3. Extend DNA strand by heating to 72oC so Taq can build the complementary strand
Repeat over and over until you have the desired amount of DNA
Gel Electrophoresis
One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis
This technique uses a gel as a molecular sieve to separate nuclei acids or proteins by
size
Restriction fragment analysis detects DNA differences that affect restriction sites
Restriction fragment analysis detects differences in the nucleotide sequences of DNA
molecules
Such analysis can rapidly provide comparative information about DNA sequences
In restriction fragment analysis, DNA fragments produced by restriction enzyme
digestion of a DNA molecule are sorted by gel electrophoresis
Restriction fragment analysis is useful for comparing two different DNA molecules,
such as two alleles for a gene
Overview of the PTC lab
Harvest your cheek cells
Boil them to rupture the cells
Add chelex to stabilize enzymes that degrade DNA
Amplify your DNA by PCR
Digest your DNA with HaeIII restriction enzymes
Run and analyze the results of gel electrophoresis
Collecting and isolating DNA
Gently scrape the inside of your cheek to harvest cells
Place into saline solution to remove contaminants
Vortex and centrifuge your tubes
Add chelex, vortex and float in boiling water
Centrifuge and place on ice
Amplifying DNA by PCR
Add loading dye to your PCR tube
Add 2.5 ul of your cheek cells
Centrifuge
Put in PCR rack
Perform PCR thermocycling
Digest DNA
Label 2 tubes
Add 10ul of your DNA to “U” tube. Put on ice
Add 15 ul DNA to “D” tube
Add 1um HaeIII to “D” tube
Centrifuge and put in warm water bath
Gel electrophoresis
Add 20 ul size marker to lane 1
Add 10 ul of “D” tube to lane 2
Add 16 ul of “U” tube to lane 3
Run R 130 V for 30 minutes
Photograph the bands
Analyze your results
Results
If your D lane has a single band you are tt, a homozygous non-taster
If your D lane has 2 bands you are TT, a homozygous taster
If your D lane has 3 bands you are Tt, a heterozygous weak taster