Thu, 30 Nov 95 07:56 +0400 MS tsai wrote to "bionet.molbio.methds-reagnts mail newsgroup" <bionet-news at daresbury.ac.uk>:
> Somebody help me !
>> I had a big trouble. Non-specific bands were presented in
> my RT-PCR products. The mRNA were purified from white blood cells
> by Pharmacia micro mRNA purification kit, first strand cDNA were
> made as the recommadation of Invitrogene cyclic CDNA systhesis
> kit by random hexmer primring. I proceeded the first PCR and nest
> PCR from ist cDNA and multiple bands wrer found. It shoud be a
> c.a. 1.2kb band only,but I could find non specific band from
> 500bp-1.5kb. I tried to raise the PCR temperature( 55,60 ,65
> degrees),decrease the Mg concentration(1.5mM, 1mM, 0.5mM) ,adjust
> the dNTP conc. ,low the random hexmers conc. in 1st strand cDNA
> synthesis ....ect. , and the results were still the same.
> Would you please tell me what's the matter with....
> Thanks !
> ps. The primers of the first and nest PCR were parts of the
> sequences of CDNA.(i.e. the exons)
Why are you sure, that your bands are nonspecific?
Do you make Southern or restriction analysis? It is possible,
that you test alternatively spliced isoforms of mRNAs or mRNAs
of strong homologous genes.
Sergey V. Orlov (serge at serge.stud.pu.ru)
IEM, dep. biochemistry
Sat, 02 Dec 95 02:20 +0400 MS