experimental methods in molecular bio

Common features of cloning vectors

• origin of replication

• selectable marker

• insertion site for cloned DNA (aka multiple cloning site/polylinker)

what strands are the forward and reverse primers added to?

Forward primer: binds to template (antisense) strand (3’ → 5’)

Reverse primer: binds to complementary (sense) strand (5’ → 3’)

what are sticky ends?

short, single-stranded DNA overhangs created by restriction enzymes that cut DNA in a staggered way

what is the purpose of sticky ends?

they base-pair with complementary sticky ends to hold the fragments together temporarily, allowing for permanent covalent bonds to form and to create recombinant DNA

importance of poly-A tail in cDNA synthesis

to act as a binding site for an oligo(dT) primer, which is necessary to initiate the reverse transcription of mRNA into cDNA

what are the two enzymatic activities of T4 DNA polymerase?

1. 5’ → 3’ polymerase activity (restores chain using dNTPs and complementary strand as template)

2. 3’ → 5’ exonuclease activity (removes nucleotides from 3’ ends of DNA)

what is bacterial transformation?

process where bacteria take up and incorporate foreign DNA into their genome