So I performed an experiment using my own human genomic DNA and PCR. The experim

Question

So I performed an experiment using my own human genomic DNA and PCR. The experiment did not work.

Could someone give me a detailed explanation as to why the PCR didn''t work?

***Some details***

Isolated Human genomic DNA in water, Human derived PCR products and Restriction Enzyme Digestion of Human derived PCR products were the 3 types of DNA loaded onto the agarose gel.

Summarized Protocol: cheek cells were obtained from the mouth and put into PBS buffer. A DNA kit containing proteinase K solution and AL solution were added. The obtained lysates were then run through nucleic acid building resin. After spinning AW1 and AW2 solutions were added. This all obtained the isolated human genomic DNA in water. 5 ul of this was then mixed wth PCR Master Mix and left to spin on a thermocycler to obtain the PCR derived prdocut. Lastly, 25 ul aliquots of RE Kas I Master Mix was added to 30 ul aliquots of the PCR product. incubation was done at 37 degrees celsius for 2.5 hours. finally with TAE buffer, 3% agarose gel and adding 2.5 ul of redsafe, each of the three samples, plus a ladder and a control were loaded onto the gel. (12 ul of DNA in water, 24 ul of PCR product and 48 ul of restriction enzyme type)

* there was vortexing and centrifugation in between all the steps

Explanation / Answer

Error in the protocol would be- use of water for keeping water. After washing with buffers AW1 and AW2, the lysates should be kept in buffer solution only. As DNA stability is disrupted due to its interaction with water.

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