Related: Restriction Cloning, ICM - Assistante Ingénieur

Designing PCR Primers

Things to consider when designing primers

18-24 base pairs
- if too short: can produce more nonspecific DNA amplification → it could produce dna that’s not what you want just because of the point where it stops accounting for unique base pairs → some of the dna products are not what you were trying to amplify
- if too long: can result in slower hybridization rates

annealing: annealing temp allows the primer to base pair with the DNA
melting: melting temps where half of the primers will dissociate from the DNA
want annealing temp to be 5 degrees below the melting
- ANNEAL = MELT - 5 degrees
- lowering the temperature even more risks nonspecific binding
the two primers (Forward/Reverse) ideally have similar melting temps
- online calculators/tools for this (thermofisher etc.)
- manually: +4 for every G or C, and +2 for every A or T
  - example primer: GCAGT AGCAT GTCAC ATATG
  - calculation: 4+4+4+2+4+2+2+4+4+2+2+4+2+4+2+4+2+2+2+2+4
  - it’s melting temp: 58 degree Celcius

idea GC content of primers are between 40-60%
GC clamp at 3’ end of primer: include 2-3 G’s/C’s if possible
GC bps are useful because they have 3H bons (vs 2H bonds for AT)
- GC pairs provide stronger binding to the template dna

References