Short PCR product

aes ali at image.dk
Tue Jan 8 06:13:59 EST 2002


That is right Duncan, thank you for the answer, I will now go ahead with the
primers. I have a side question that you might have an opinion on!

I am going to attempt to do quantitative PCR with degenerate primers, I can
make the primers so that they should amplify all the genes I want, but again
the primers end up being 28 bp long with 8 bases (in the middle) having
degenerate composition (R,K and Y). Do you think it is possible to get these
primers working in a semi quantitative experiment?

Thank you

Aes
"Duncan Clark" <junk@[127.0.0.1]> wrote in message
news:xZlxXY8TwrO8EA+T at genesys.demon.co.uk...
> In message
> <Pine.GSO.4.31.0201071243480.19893-100000 at ascc.artsci.wustl.edu>, Alex
> Brands <abbrands at artsci.wustl.edu> writes on the Mon, 7 Jan 2002
> >On Sat, 5 Jan 2002, aes wrote:
> >
> >> Hi.
> >> I am going to run PCR with 35 bp long primers, with an amplicon in
between
> >> of 15 bp, so that the total lenght of the PCR product is 85 bp (for
> >> quantitation by real time pcr and sybr green1). My question is, what is
the
> >> shortest posible amplicon? And is 15 bp to short?
> >> Thank you and happy new year
> >
> >Have you considered synthesizing the amplicon?  you can design a handful
> >of overlapping oligos like this:
> >
> >---------  ---------  ----------  -----------
> >-------------  ------------  -------  -------
> >
> >Kinase the oligos, mix them all together and treat with ligase.
> >
> >
>
> But he's not trying to do gene synthesis.
>
> He is running quantitative real-time PCR of an 85bp amplicon.
>
> Duncan
> --
> The problem with being on the cutting edge is that you occasionally get
> sliced from time to time....
>
> Duncan Clark
> GeneSys Ltd.





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