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Polymerase chain reaction

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Mary set up a similar PCR reaction. The Tm of the forward and reverse primers she uses is 55oC. The conditions that were optimized by her instructor were as follows:

Cycle
Temperature (oC)
Time

1X
94o
3 minutes

30X
94o
30 seconds
Denaturation
50o
30 seconds
Annealing
72o
60 seconds
Extension

Mary sets up the PCR, but decides to see if she can get better results than her instructor, and makes the following changes in separate PCR runs.

Describe the effect that each of her changes would have on the amount and purity of her PCR product. (Briefly explain).

A.She changes the annealing temperature to 60oC

B.She increases the number of cycles to 31

C.She changes the magnesium concentration from 1.0 mM to 2.0 mM

D.She adds non-complementary restriction enzyme sequences to the 3' ends of the primers

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Mary set up a similar PCR reaction. The Tm of the forward and reverse primers she uses is 55oC. The conditions that were optimized by her instructor were as follows:

Cycle
Temperature (oC)
Time

1X
94o
3 minutes

30X
94o
30 seconds
Denaturation
50o
30 seconds
Annealing
72o
60 seconds
Extension

Mary sets up the PCR, but decides to see if she can get better results than her instructor, and makes the following changes in separate PCR runs.

Describe the effect that each of her changes would have on the amount and purity of her PCR product. (Briefly explain).

A.She changes the annealing temperature to ...

Solution Summary

Answers to several questions regarding PCR are provided.

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