You might actually have too much template to start with. You can try
either dilute your fisrt RT-PCR product (10 up to one million times) or
try lower PCR cycles numbers;
Hope this helps.
Dr O. Gandrillon
Département de biologie
Ecole Normale supérieure de Lyon
46, allée d'Italie
69364 Lyon Cedex 7
E.mail: ogandril at ens-lyon.fr
"The reasonable man adapts himself to the world; the unreasonable one
persists in trying to adapt the world to himself. Therefore, all
progress depends on the unreasonable man."
George Bernard Shaw
tahar aboulkassim wrote:
>> Hi everyone!
> I did an RT-PCR and I had a band with the right size. I purified this band
> from the gel and dissolve it in tris buffer. then I used 1µl of this RT-PCR
> product and run another PCR (All the parameters were the same indeed the
> primers).But I always obtain a smear.
> I have no explanation for that and I will be so grateful if someone gives me
> an idea.
>> thank you so much for help.
>> A. Taha
> E- mail : ellape at sprint.ca