2 ObjectiveReview DNA replication, knowledge of the process, and how it occurs.The concept of PCRThe purpose of DNA and RNA extraction relative to PCR.The process of PCRthe purpose of a primerBrief background of history of taq and its purpose in PCR
3 DNA replicationReplication allows our genetic material to be passed on to daughter cells.
12 Thermal cycler Machine used for PCR amplification Raises and lower temperatures for temperature sensitive reactions
13 Steps for PCR Step 1: Denaturing Heat 94-98°C Causes the DNA to separate by disrupting the hydrogen bonds between strands
14 Steps for PCR Step 2: Annealing Lower temperature to 50-65°C Allows primer to bind to DNA strand – H bonding between DNA and primersOne primer binds to 5’ end and one to 3’ end
15 Steps for PCR Step 3: Elongation Temperature depends on polymerase you usetaq DNA polymerase works best at 75-80°CPolymerase add dNTPs to primer and extends the strand.
16 The history of taqHeating inactivates the DNA polymerase used before taq was discovered Researchers needed a DNA polymerase that didn’t need replacing after every round of PCRtaq DNA polymerase was discovered in bacteria called Thermus aquaticus that lived in underwater hydrothermal vents
21 Gel Electrophoresis Run product on an agarose gel Electric current is run through the gel to separate product based on sizeEnsures the correct product was amplified
22 Gel electrophoresisLadder – DNA solution of varying sizes and used to compare samplesPositive control – a group where you expect an outcomeNegative control – a group where you expect nothing to happenLarger fragments are closer to the top