AGAROSE GEL ELECTROPHORESIS AND PCR
Agarose gel electrophoresis
Polymerase chain reactions
Function
Obtained the size of DNA sample
Separate DNA fragment
Agarose as matrix
Need current
Running buffer/electrophoresis buffer
Buffer in electrophoresis tank
Agarose is dissolves in running buffer
Buffer
1x TAE (tris-acetic acid-EDTA)
1x TBE (Tris-boric acid-EDTA)
DNA ladder/DNA marker
Ruler to indicate the size of the separated DNA fragments.
Range of DNA ladder used must serve the range of DNA fragments separated.
Apply in one of the wells in agarose gel prior electrophoresis.
DNA sample mixed with DNA loading dye before applied into the well.
DNA loading dye
Purpose
Provide density to DNA
Serves as tracking dye in monitoring fragments of DNA movement in the gel.
Each colour represent specific molecular weight of DNA.
Application voltage is 1-5 volt/ centimetre of agarose
Ethidium bromide
Bind to the backbone of DNA
Gives fluorescence orange under UV light
Function
Amplify specific DNA region through imitation of DNA replication process inside cell.
Required of PCR reaction
Mg2+
DNA polymerase buffer
A pair of primer
DNA template
Taq DNA polymerase
Preparation of PCR mix carried out on ice.
PCR cycle
Anneal
Extend
Denature
At 94˚C for 20 seconds
Hydrogen bonding between nucleotides break .
At 55˚C for 20 seconds
A pair of primer anneal to the complementary sequence of DNA template.
At 72˚C for 30 seconds
DNA polymerase elongates the synthesis of new DNA strands .