1.3 PCR rxn

Question 1

list the five components used in PCR

Answer 1

1. buffer solution
2. DNA extracted from soil sample
3. E. coli primers
4. free DNA nucleotides of each of the 4 bases
5. DNA polymerase (Taq polymerase – a heat tolerant enzyme that ca survive high temperatures used in PCR)

Question 2

1st step

Answer 2

add all solutions to reaction tube and mix. – after mixing place tube in the thermocycler

– assuming the sample contains E. coli DNA, the primers we include target a particular region of E. coli for DNA amplification

Question 3

2nd step (cycle begins) – first step of CYCLE

Answer 3

each cycle begins with the temperature increased to 94ºC – this denatures the DNA into single strands.

Question 4

3rd step – second step of CYCLE

Answer 4

the temperature is lowered to 55ºC to allow primers to bind/ANNEAL to respective template strands. – the primers are only complementary to the 3’ ends of the target region.

Question 5

if target region is not present in the sample

Answer 5

the primers will not bind and nothing will be amplified.

Question 6

4th step – third step of CYCLE

Answer 6

the thermocycler then raises the temperature to 72ºC which is the operating temperature for the Taq DNA polymerase.
polymerase will bind to the 3’ ends of the primers. – using the free nucleotides present in the reaction mixture, the polymerase extends ew DNA strands from each primer – new strand is synthesized. this completes 1 PCR cycle

Question 7

after how many cycles is the PCR reaction complete

Answer 7

30 cycles. – you can use agarose gel electrophoresis to visualize the PCR product produced.