PCR and primer design

Question 1

Does the taq polymerase generate any overhang?

Answer 1

Yes. 3’ A/AA overhang.

Question 2

What does the primers’ melting temperature depend on?

Answer 2

Ions, solvent, primer length, GC-content.

Question 3

What affects primer stability and efficiency?

Answer 3

The following attributes are positive:
1. Primer 3’ stability
2. No simple regions (repetative)
3. No 3’ complementarity between the primers.
4. Tm for both primers match within 5 degrees.