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RDT Practical

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Boka Pola
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7 views2 pages

RDT Practical

Uploaded by

Boka Pola
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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← For 30 cycles →

Analysis on Agarose Gel

4. Following PCR amplification, add 5 µl of Gel loading buffer to each of the PCR tubes.
5. Tap the mixture thoroughly and wait for a few seconds for the 2 layers to separate.
6. Carefully pipette out 15 ul of reaction mixture (avoiding mineral oil layer) and load in
the well of the 1.5% agarose gel.
7. Load 10 µl of the ready to use marker provided. Note down the order in which the
samples have been loaded.
8. Electrophorese the samples at 100 volts for 1-2 hours till the tracking dye
(bromophenol blue) reaches 3 / (4 ^ m) of the length of the gel.
9. Visualize the gel under a UV transilluminator.

Observation:

Compare the amplified band with StepUp™ 100 bp DNA ladder and note down the size of
the fragment. Also observe for presence of any other bands apart from the amplified product.

Lane 1: StepUp™™ 100 bp DNA Ladder (100 bp to 1000 bp).

Lane 2: PCR Amplified 0.8 kb fragment electrophoresing along- side 0.8 kb fragment of
StepUp™ 100 bp DNA ladder.

Fig 1: PCR amplified product electrophoresed on a 1.5% Agarose gel (Stained with
EtBr)

Interpretation:As observed on agarose gel, PCR amplification of the template using specific
primers results in a specific product of a particular length. The conditions have been
optimised to give a highly specific product of 800 bp as is observed by the absence of any
non-specific products. Altering these conditions would result
in non-specific amplification.

PCR was carried out with 100 nanograms of template,


having approximately 1000 copies of the target sequence.
Following PCR, the product yield is in microgram quantity,
which is approximately a million copies of the target
sequence, highlighting the fact that PCR is a very sensitive
technique.

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14. Then load the sample in different lanes:
1st lane – EcoRI digested Solution
2nd lane – Hind III digested solution
3rd lane – Controlled DNA
4th lane – Marker Sample
15. Start electrophoresis: switch off when tracking dye (bromophenol blue) reaches 3/4th of
the gel from the wells
16. Then DNA samples can be visualized under UV light

Observation: observe the DNA fragment obtained on restriction digestion with EcoRI and
Hind III. Compare this with molecular weight maker (λ 1ml uI digest)

Conclusion: restriction patterns obtained on digestion with EcoRI and Hind III are markedly
different, demonstrating the fact that each restriction enzyme recognizes and cleaves only a
particular base sequence unique to it.

By comparing the migration distance of the digested DNA fragments with


that of the marker. One can also determine the approximate size of DNA fragments.

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