PCR And qPCR Flashcards
What does pcr need
Taq polymerase
What type of reaction is a pcr and why
Chain reaction as the dna duplex formed at the end of the reaction is used as a template for the next reaction
What is the definition of pcr
Lab technique that allows rapid amplification of a specific dna segment or gene
What are the 3 steps in pcr
- denaturing: dna strands separate
- annealing: primers adhere to dna strands
- extension: Taq polymerase builds complementary strand
What temp do each steps of pcr have
- denaturing: 95 degrees
- annealing: 55 degrees
- extension: 72 degrees
How long does pcr take for 25-30 cycles
45mins - 1hr
What can make make the time pcr takes change
- how many cycles needed
- the dna template
- enzymes used
What can make make the time pcr takes change
- how many cycles needed
- the dna template
- enzymes used
What are the components of pcr
- dNTPs
- reaction buffer
- primers
- DNA template
- Taq polymerase enzyme
What are dNTPs
- guanine (dGTP)
- cytosine (dCTP)
- thymine (dTTP)
- adenine (dATP)
What is Taq polymerase
Thermostable dna polymerase 1
What are primers
- Short, single stranded segments of dna that are complementary to the beginning and end of dna sequence
What are the 2 types of primers
- forward: binds to antisense strand that moves from 3” to 5”
- amplification of antisense strand
- reverse: binds to sense strand that moves from 5” to 3”
- amplification of sense strand
What is a reaction buffer
- provides suitable chemical environment for activity of Taq polymerase
- buffer ph of 8-9.5
- magnesium ions are essential cofactors for polymerase activity and help primers bind
What are thermocyclers
Regulate temperatures in a cyclical program
How can we visualise pcr amplified dna
Agarose gel electrophoresis
What does agarose gel electrophoresis do
Visualise different size of dna regions that are amplified
