pcr from plant tissues

Jesse J. Parry casshan at siu.edu
Sat Jun 27 21:04:55 EST 1998


Jimmy Loh wrote in message ...
>Hi netters!
>
>I'm having problems doing PCR on my potential transgenic plants.
>Means of transformation is via gene gun  bombardment.
>
>DNA was extracted by using DNAzol or other conventional means and
>run on a gel to check. Seems ok.
>
>What I am wondering is...whether the PCR conditions should be
>varied. Perhaps the gene is in such low concentrations that PCR
>can't pick it up? Or are there special tricks of the trade to
>enhance PCR from plant tissues? SUch as..i dunno...adding
>DMSO or whatever. I run a normal PCR on the gene from the plasmid
>as an additional postive control for the pcr and it works..so the
>problem shouldn't be in the PCR reagents.
>
>Any suggestions are greatly appreciated.
>
>Jimmy Loh
>NTU-NIE
>Singapore

>

Jimmy, did you quantify your DNA by spectroscopy before running on the gel?
Also, did you add RNase to the resuspended DNA to ensure that you were
getting the right nucleic acid?  My advice to you is to develop an internal
control for the sequence you wish to amplify (that is, of course, if you
know the sequence) as a means to compare relative intensities of different
sized fragments.  Also, you need to consider how much protein and sugar may
be present in your sample, because excess polysaccharides can inhibit
extraction of DNA.  You also need to take into consideration from the type
of tissue you are extracting.  Seed extraction can be a problem because
bombardment of plant tissue can lead to numerous copies of the sequence
being present in the seed coat.  If you have any further questions, please
feel free to e-mail me at casshan at siu.edu

Jesse Parry






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