DNA techniques (PCR)

PCR reaction requirements

DNA polymerase, nucleotides, DNA, primers specific to fragment desired to replicate, polymerase cofactor (Mg2+)

denature

(95-98 C) separates DNA strands by denaturing H-bonds

anneal

(50-60 C) allows primers to bond (anneal) to their complementary sequences

extend

(72 C) DNA pol. binds to primers and extends (synthesizes DNA)

gel electrophoresis

separates DNA by size, DNA is loaded into wells of agarose gel and ran through electricity, gel is stained and viewed under UV light

restriction enzymes

cut DNA at a specific sequence

RE uses

RFLP, cloning DNA fragments into plasmids (recombinant DNA)

plasmids

small, circular, extrachromosomal piece of DNA derived from bacteria

DNA ligase is used to

connect backbones of sticky DNA pieces