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Thick 100-bp band disappeared under UV - (May/21/2014 )

Hi,

 

I used "Alkaline Lysis Method" to extract a 4-kb plasmid from E. coli. I then single-digested the plasmid with a restriction enzyme and ran it on an agarose gel. Surprisingly:

 

1) Before the digestion, my evaluation of the DNA concentration turned out to be huge (5000 ng/ul)

2) On the gel: I got a very thick band around 100 bp size, beside the 4-kb plasmid.

3) The 100-bp band gradually disappeared when I left the UV on for 10 min.

 

I assume:

1) The 100-bp band was RNA due to inefficient function of RNAse-A during the miniprep procedure.

2) They got all digested by the restriction enzyme to 100 bp fragments and stuck-up to form thick bands.

3) Under the UV, the RNA degraded.

 

In your opinion, am I right?

Are there other explanations??

 

I thank you in advance for your responses.

-IPI-

Restriction enzymes don't digest RNA!  The band could be degraded DNA or RNA, both will appear as a smear of relatively small size. 

 

It is extremely unusual to get 5000 ng/ul of DNA out for a pure plasmid extraction, there is a good chance that your plasmid is contaminated with either degraded DNA, intact genomic DNA, or RNA.

 

Leaving the UV on for 10 min is irrelevant, any DNA will be damaged by this length of exposure - 10 seconds is enough to damage most DNA.

-bob1-

could there be a combination of diffusion and bleaching of the 100 bp band to cause its apparent disappearance?

-mdfenko-

It is extremely unusual to get 5000 ng/ul of DNA out for a pure plasmid extraction, there is a good chance that your plasmid is contaminated with either degraded DNA, intact genomic DNA, or RNA.


Also it's extremely unusual to be able to get a measurement of 5 ug/ul concentration. Or more like not possible, since it lies outside the measurable range. If the machine gave this out this reading, I would consider it anything but the real concentration (including machine error and little frog siting inside the cuvette).

-Trof-