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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