End of PCR

Question 1

What do primers determine?

Answer 1

The specificity of amplification

Question 2

What do we consider when we design primers?

Answer 2

18-24 nt in length and primer pair cannpt differ by >3nt
G/C content is 40-60% with no long run of bases and G/C is at the 3’OH end
The calculated Tm of primer shouldn’t differ by more thab >5 degrees

Question 3

What are the other two players in PCR

Answer 3

magnesium ions
Nucelotides

Question 4

If the temp is too low what will happen to the denaturing of the DNA

Answer 4

only the AT bonds will denature

Question 5

What are some thermocycling parameters?

Answer 5

Denaturing - Temp must be high enpugh to ensure full denaturation (GC and AT)
Annealing - too high - no annelaing
too low - non-specific amplification
Extension - must be near the optimum temp for the polymerase (74)