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....
ps. The primers of the first and nest PCR were parts of the
sequences of CDNA.(i.e. the exons)