1,000,000 1,000,000,000 PCR cycles 20 40 30 Theorectical yield Actual yield “Plateau effect” No....
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Transcript of 1,000,000 1,000,000,000 PCR cycles 20 40 30 Theorectical yield Actual yield “Plateau effect” No....
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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?
![Page 2: 1,000,000 1,000,000,000 PCR cycles 20 40 30 Theorectical yield Actual yield “Plateau effect” No. of DNA copies How much DNA amplification has occurred?](https://reader036.fdocuments.us/reader036/viewer/2022081519/56649d825503460f94a67bea/html5/thumbnails/2.jpg)
How do scientists
analyse these PCR products?
![Page 3: 1,000,000 1,000,000,000 PCR cycles 20 40 30 Theorectical yield Actual yield “Plateau effect” No. of DNA copies How much DNA amplification has occurred?](https://reader036.fdocuments.us/reader036/viewer/2022081519/56649d825503460f94a67bea/html5/thumbnails/3.jpg)
Agarose Gel Electrophoresis
Separates DNA fragments according to
their size
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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
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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
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Agarose gel
Loading DNA samples
![Page 7: 1,000,000 1,000,000,000 PCR cycles 20 40 30 Theorectical yield Actual yield “Plateau effect” No. of DNA copies How much DNA amplification has occurred?](https://reader036.fdocuments.us/reader036/viewer/2022081519/56649d825503460f94a67bea/html5/thumbnails/7.jpg)
Agarose gel
Loading DNA samples
![Page 8: 1,000,000 1,000,000,000 PCR cycles 20 40 30 Theorectical yield Actual yield “Plateau effect” No. of DNA copies How much DNA amplification has occurred?](https://reader036.fdocuments.us/reader036/viewer/2022081519/56649d825503460f94a67bea/html5/thumbnails/8.jpg)
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”
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Separating the DNA Fragments
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Separating the DNA Fragments
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Separating the DNA Fragments
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Visualising DNA
The UV light excites the DNA SafeView (fluorophore) that is bound to the DNA
Transilluminator