PCR problem with EGFP

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Tue Sep 21 06:32:03 EST 1999



Björn Fritz wrote:

> Hi all,
>
> I have severe problems in doing a PCR with a plasmid, that contains
> GFP.  The primers I designed prime in the enhanced GFP gene. I used 4
> different primers, checked them with several primer design programs,
> used different temperatures, polymerases and buffers, checked the
> quality of the template. I am quite sure that the template is okay
> because I used as well the original plasmid I got from Clontech. But
> all primers don´t work even on the plasmid. Another group working for
> years with the same plasmid states that it has the same problems.
> Clontech has no idea.
> Does anyone of you has  primersequences in the EGFP that work well? I
> would be very glad for any suggestions
>
> Thank you

Hi Björn,
Unfortunately you do not tell us what kind of problem you have. Is there
no product at all, or do you get multiple bands, or smear? If you have
multiple bands or a smear, I guess you use too much template. PCR
usually works best with low amounts of template. In case of a plasmid
template, I use 1ng routinely. In case you have no product at all, your
dNTPs might have gone off. That sometimes happens, especially with cheap
nucleotides or after multiple freeze-thaw cycles. Use fresh dNTPs.


Hope this helps,
Frank

PS: I haven´t had any problems amplifying a plasmid containing the EGFP
gene. However, in this case the total plasmid was amplified, not only
the EGFP coding region.





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