non specific bands of RT-PCR

tsai n20248 at vghtc.vghtc.gov.tw
Wed Nov 29 22:56:33 EST 1995


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)




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