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AGAROSE GEL ELECTROPHORESIS & PCR : (Agarose gel electrophorosis…
AGAROSE GEL ELECTROPHORESIS & PCR :
Polymerase Chain Reactions
to amplify specific DNA region ( fragment) through imitation of DNA replication process inside cell
Requirement of PCR reactions (PCR mix):
DNA template
DNA polymerase buffer
Taq DNA polymerase
Sterile dH20 to make up the desired volume of reaction
preparation of PCR mix is carried out on ice
PCR steps:
Anneal at 55 degree celcius for 20 sec
a pair of primers ( forward & reverse) anneal to the complementary sequnece on DNA template
Extend at 72 degree celcius for 30 sec
DNA polymerase elongates the synthesis of new DNA strands in the direction 5' to 3'
denature at 94 degree celcius for 20 sec
hydrogen bonding between nucleotides break resulting ssDNA
DNA ladder/ DNA marker
Range of DNA ladder must serve the range of DNA fragments seperated
DNA sample must be mixed with DNA loading dye before applied into the well
DNA ladder serve as ruler to indicate the size of seperated DNA fragments
Example of DNA markers
DNA ladder/ DNA marker will applied in one of the wells in agarose gel prior electrophoresis
DNA loading dye
purpose of DNA loading dye
serves as tracking dye in monitoring fragments of DNA movement in the gel
colour band in tracking dye represent specific molecular weight of DNA
application of voltage is 1 - 5 volt/ centimetre of agarose
seperated DNA fragments can be viewed under UV light
Ethidium bromide will bind to the backbone of DNA and give fluorescence orange under UV light
DNA ladder and DNA sample is labelled using Alpha Imager software before printing
AGE results always reported in the format of black and white print-out
provide density to DNA so that it sink the agarose well
example of loading dyes
Agarose gel electrophorosis
percentage/ concentration of agarose will predetermined the optimum DNA fragments seperation
running buffer/electrophoresis play important role in preparation of agarose gel and also in electrophoresis tank.
type of buffer
1 x TAE ( Tris- acetic acid - EDTA)
1 X TBE ( Tris-boric acid -EDTA)
matrix is used to seperate DNA fragments by application of current
the size of DNA sample can be estimated using AGE approach
ethidium bromide must be added to completely dissolved buffer
