st2153 at zi.biologie.uni-muenchen.de
Fri Mar 26 08:14:43 EST 1999
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
thank you so much for help.
E- mail : ellape at sprint.ca
I had similar problems with normal (non-RT) PCR. Finally I got convinced
that the second amplification round amplifies artefacts preferiantilly.
This might be because these artefacts arise in (nearly) every PCR reaction
but in low amounts so you wont see them on the gel. (People that amplify
elecronic signals observe similar problems: the more you amplify a signal
the more backround will arise - not linearily)
If you know the product you are looking for you should use nested primers.
I got rid of the smear and the artefacts using one nested primer and
dilute the first amplificate 1/1000 or more - and not more than 25 cycles.
If you use nested primer you need not purify the first PCR on the gel
because only the wanted product will amplify.
hope this helps
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