Primer design

Question 1

what size must primers be?

Answer 1

must be >= 18 nt long - gives more specificity - only binds to gene of interest

Question 2

why must primer end at 3’ end?

Answer 2

elongation occurs here - end in C or G (3H bonds firmly hold primer to genomic DNA)

Question 3

what must the Tm (melting temp) be?

Answer 3

values of primer pair must be within 5 degrees of each other

Question 4

what must the annealing temperature be for PCR?

Answer 4

5 degrees less than lowest Tm Value - if too high = no sticking
too low = sticking anywhere (lose specificity)

Question 5

what direction will DNA only be synthesised in?

Answer 5

5’-3’

ssDNA only hybridise/anneal in antiparallel form

Question 6

When calculating Tm, what must be removed when counting bases?

Answer 6

restriction endonucleases

Question 7

what must also be considered when calculating Tm?

Answer 7

nucleotide length, ensure not to count more C/G than necessary

Question 8

how do you calculate the annealing temperature?

Answer 8

Tm - 5

Question 9

what would make you decide which pET vector to use for cloning?

Answer 9

if the His-tag was coded for downstream and the gene has no stop codon