RT-PCR and plant transformations

Mike Prigge prigge at darkwing.uoregon.edu
Mon May 10 23:19:41 EST 1999


In article <199905100106_MC2-7511-ECED at compuserve.com>,
105012.2233 at COMPUSERVE.COM ("Engelina S. Olsthoorn") wrote:

> 
> Questions: 
> 1. (Glycine max L. cv. Kitano-shiki). What does that mean?

Kitano-shiki is a particular strain of soybean (Glycine max).
  
> According to "Molecular Biomethods Handbook" by Rapley/Walker (page 311) 
> the RNA-PCR  modification of the original PCR procedure may be used
> to amplify RNA species. This requires the production of complementary
> DNA (cDNA) to the target RNA sequence. This is undertaken using an 
> appropriate oligotide primer, dNTPs, and the enzyme reverse 
>  transcriptase. The resulting DNA may then be used in the PCR directly.
>
> 2.  What is the function of the polyCAT mRNA?
> Also they already had ferritin cDNA, so why use RT-PCR?
> They can use random primers????? What does 'random primer p[dN]6)' mean?

Is it possible that it says 'poly(A)+'?  Purifying poly(A)+ RNA is a way
to get rid of most of the tRNA and rRNA (which comprises most of the total
RNA) leaving predominantly mRNA.   'Random primer p[dN]6' means
5'-NNNNNN-3' where N is any deoxynucleotide.  During the RT step, one can
use random primers, poly(dT) primers, or gene-specific primers.  It really
doesn't matter as long as you use at least one gene-specific primer during
the PCR step.  And, as for why use RT-PCR instead of ordering the
previously-isolated cDNA from whomever isolated it... these days it is
often faster just to isolate something yourself instead of waiting for the
person to send it.  If all goes well you can have it subcloned in a couple
days (especially if you already had poly(A)+ RNA already).

> 3. The pCR II vector, is that a bacteriophage?

It is a plasmid (actually a souped-up kind of plasmid called a 'phagemid')
that is optimized for cloning PCR products.  (Phagemids allow one to
purify single-stranded copies that can be used for sequencing.)

> 4. Where can I read more about RT-PCR?
> Where can I read more about the basics of agrobacterium-mediated plant
> transformation?

Agrobacteria info is found in many Genetics textbooks (eg, Snustad et al
and Suzuki et al. and Genes V).  For RT-PCR, you'll probably have to find
a book on PCR from your library or do an internet search.

> Your help will be much appreciated!!! Thank you.

good luck, 
Mike
-- 
Michael Prigge                          Phone: (541) 346-4256
Institute of Molecular Biology            Fax: (541) 346-5891
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