1,000,000 1,000,000,000 pcr cycles 20 40 30 theorectical yield actual yield “plateau effect” no....
TRANSCRIPT
1,000,000
1,000,000,000
PCR cycles20 4030
Theorecticalyield
Actual yield“Plateau effect”
No.
of
DN
A c
opie
sHow much DNA
amplification has occurred?
How do scientists
analyse these PCR products?
Agarose Gel Electrophoresis
Separates DNA fragments according to
their size
Step 1 – Agarose is added to buffer and heated to 95ºC (to melt it). After cooling, it is poured into a pre-preparedcasting tray
Step 2 – a comb is inserted immediately to create wellsfor loading the samples
Step 3 – the gel is left to set
Step 4 – the comb is removed slowly(see set of wells that have been created)
Making Agarose Gel
Agarose gel
Well 1 Well 2 Well 3 Well 4 Well 5
Making Agarose Gel
A fluorophore (DNA SafeView) is added to the gel. This intercalates with the DNA & fluoresces when
excited by UV light
Agarose gel
Loading DNA samples
Agarose gel
Loading DNA samples
Separating the DNA Fragments
After the samples are loaded into the wells, an electric current is applied across the gel and the gel is “run”
__ ++
Separating the DNA Fragments
__ ++
Separating the DNA Fragments
__ ++
Separating the DNA Fragments
Visualising DNA
The UV light excites the DNA SafeView (fluorophore) that is bound to the DNA
Transilluminator