Name 2 factors that affect the melting temperature (Tm) of a primer used in PCR. What step in the PCR reaction will be affected most by the Tm of the primers? What will happen if the temperature of this step is too high? too low?

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Irving HeathcoteLv2

28 Sep 2019

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Related questions

a) What would be the expected outcome if the annealing temperature of a PCR was too high for the primers in the reaction?

b)What would be the expected outcome if the annealing temperature of a PCR was too low for the primers in the reaction?

Please answer the following genetics questions pertaining to primers:

1.) What is the effect of primer length on a PCR reaction? Please fill in the blank for both (a) and (b) below:

a. Shorter than 18-20 bp: ________________________

b. Longer than 18-20 bp: ________________________

2.) How would you rate efficiency of a primer pair if the difference in Tm of the forward and reverse primers is more than 5Â°C apart?

3.) Define the problem of âprimer self-complementarity.â Also describe âprimer loopâ and âprimer dimerâ phenomena in the PCR reaction.

orangegnat260

1. During which step(s) of the PCR cycle are phosphodiester bonds being formed?
a. Denaturation
b. Annealing
c. Extension
d. During both annealing and extension

2. Estimate the Tm of the following primer using the method in this handout. (One easy method to
estimate the Tm of a primer is to count the number of G and C residues and multiply that
by 4Â°C then count the number of A and T residues and multiply that by 2Â°C then add the
two numbers.)
5â TTATGCATGACTAATAGAA 3â

3. How many Âµl of the pGLO plasmid will you need for a PCR reaction if you need 500ng and the plasmid concentration is 0.05Âµg/Âµl?

4. What will the final concentration of the primer be if you add 1 Âµl of a 10ÂµM stock in a reaction with a final volume of 25 Âµl?