In article <5teJ2.35449$Mb.17660613 at newscontent-02.sprint.ca>,
"tahar aboulkassim" <ellape at sprint.ca> 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>>Hi,
I do this experiment in my lab, last year.
I think, i don't understand why you do a second pcr, if you have the band at
the right size, but i you want to do this, you must dilute the rt-pcr
product, because this solution contain a lot of copy of your band. (dilution
1/1000 or more).
Attention at your purification, it's important that you cut only the right
band otherwise you can have pblms with other product. (Perhaps you can try a
electrophoresis with a gel more concentrated or two migration).
I think you need reduce the cycle number in the PCR (only 10 or 15 cycles).
I hope this helps. (if you have some question, don't hesitate to write me)
PS: i sorry for my bad english !!!!
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