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Extracting Plasmid DNA
Name: Ashley
Status: Student
Age: 18
Location: N/A
Country: N/A
Date: 2001
Question:
When conducting an experiment where you extract plasmid
DNA from a cell and insert it into an ampicillian resistant cell, and if
we kept the experiment to extract the plasmid the same what would happen
is RNA was the transforming principle and not DNA? would you get the same
result?
Replies:
Dear Ashley,
The answer to your question depends upon the purity of your extracted
plasmid DNA. If you're certain that there is little or no RNA contamination
of your plasmid DNA extract, then you would not expect to obtain any
ampicillin-resistant transformants if RNA were actually the transforming
principle. This level of purity would probably be most easily attained by
RNase digestion of the plasmid DNA prior to transformation to degrade
residual RNA.
If your plasmid extract contains a mixture of DNA & RNA, you wouldn't be
able to determine which one is actually the transforming principle. But if
you were to obtain ampicillin-resistant transformants from a DNA/RNA mixture
that had been digested with DNase to degrade the DNA, it would be a good
indication that the RNA was actually the transforming principle. Bear in
mind that these experiments alone would not be definitive, but could be used
to design follow-up studies to determine whether DNA or RNA was actually the
transforming principle.
Jeff Buzby, Ph.D.
Dear Ashley,
Your question is not very clear. If your plasmid
contains an ampicillan resistance gene, you can not
select for transformants when you transform it into
cells already resistant. Is that your question? The
protocol for extracting plasmid DNA is not the same
as that for extracting RNA. You can not transform
bacteria with RNA (as far as I know), even if you were
able to get the RNA in the cell, it could not
replicate (unless it is an RNA virus) nor could it
recombine into the DNA of the cell.
If this does not answer your question, please
rephrase.
Trudy Wassenaar
Let us back up a little. In the first experiment, you are putting DNA that
contains at least two important genes into ampicillan-SENSITIVE cells. The
first gene (_ori_) is the origin of replication, that allows the plasmid to
replicate in the cells, and the second is the ampicillan-resistance gene so
that the transformed cells can be selected for on media containing
ampicillan.
So, if you attempted to transform with RNA, you would run into (at least) one
major problem: How to have the RNA get changed back into DNA so it could
replicate and be present in all the succeeding generations. After all, even
if you could get RNA with the above two genes inserted into a cell, that is
only ONE cell, not the millions you want to grow up.
All that being said, I think it is a moot point; I know of no successful
attempts to transform using RNA. While some types of cells contain enzymes
that can convert RNA to DNA, bacteria don't appear to possess these. Hope
this helps.
Paul Mahoney, Ph.D.
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Update: June 2012
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