Past exam knowledge

Question 1

what is Gel electrophoresis?

Answer 1

a technique used to separate DNA fragments according to their size. DNA samples are loaded into wells (indentations) at one end of a gel, and an electric current is applied to pull them through the gel.

Question 2

why do DNA fragments move to the positively charged end of the electrophoresis gel?

Answer 2

DNA fragments are negatively charged, so they move towards the positive electrode

Question 3

What are the rules for designing a primer?

Answer 3

Rule 1: Primers should be 17-28 bases in length;

Rule 2: Base composition should be 50-60% (G+C);

Rule 3: Tms between 55-70 ˚C are preferred;

Rule 4: Primers should end (3’) in a G or C, or CG or GC: this prevents “breathing” of ends and increases efficiency of priming;

Rule 5: Runs of three or more Cs or Gs at the 3’-ends of primers may promote mispriming at G or C-rich sequences (because of stability of annealing), and should be avoided;

Rule 6: The 3’-ends of a pair of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesised preferentially to any other product;

Rule 6: Primer self-complementarity (ability to form 2 ˚ structures such as hairpins) should be avoided. (same is forward and reverse direction)