PCR

Question 1

What are the three steps of PCR? (3)

Answer 1

1. Denaturation
2. Primer Annealing
3. Amplification

Question 2

What are the two types of primers? (2)

Answer 2

• Custom primers = Used to amplify a sequence of interest

- Universal primers = NOT custom. Available to multiple people

Question 3

What are the precaution and drawbacks of PCR? (3)

Answer 3

• Amplifying the wrong sequence = Primers anneal to the wrong target resolved using a hot-start polymerase. Need to confirm identity of PCR products
• Contamination. Set up PCR in different area, use -ve control to check against
• Polymerase error rates. Mistakes made in rep

Question 4

What are the X and Y axes titles in a standard curve of a real time PCR experiment?

Answer 4

• X = # of templates per reaction

- Y = Crossing Point, unit = CP

Question 5

How high do the X and Y axes go on a standard curve in a real time PCR experiment?

Answer 5

• X = 40

- Y = 1.00E+10

Question 6

What direction does the graph go in a standard curve real time PCR experiment?

Answer 6

• Down/Decreases

Question 7

In a real time experiment graph what are the x and y axes and what are the max values?

Answer 7

• X = Cycle Number, max = 45

- Y = Fluorescence, max = 5.5