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From: "TOM W. QUINN" <tquinndu.edu>
Message-Id: <199510111742.LAA30256phoebe.cair.du.edu>
Subject: Re: ANimal GENe MAPpers Discussion Group
To: angenmapiastate.edu (angenmap)
Date: Wed, 11 Oct 1995 11:42:40 -0600 (MDT)
In-Reply-To: <9510111620.AA10901pv6f01.vincent.iastate.edu> from "angenmap" at Oct 11, 95 11:20:33 am
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> From: Steve Kemp <S.J.Kempliverpool.ac.uk>
> Subject: Re: ANimal GENe MAPpers Discussion Group
>
> Does anyone have a simple method for crude DNA preparations from
> cryoprserved bovine semen ? I have tried chelex and NaOH lysis and both fail
> to produce amplifiable DNA. If I follow these with phenol extraction and
> precipitation it amplifies OK, but this is too slow for large numbers of
> samples. There seems to be something in the semen or the cryopreservation
> medium which inhibits taq polymerase. Has anyone any ideas ?
>
> Thanks
>
> Steve Kemp
> - ---------------------------------------
> Stephen J Kemp
> Department of Genetics & Microbiology
> University of Liverpool
> Liverpool L69 3BX
> UK
> Tel: 0151-794-3618/3622
> Fax: 0151-794-3655
> email: KEMPSJLIV.AC.UK
> - ---------------------------------------
>
Steve: You may have already tried these, but in ancient DNA work (and with
problematic modern templates) the addition of BSA often helps to mop up
inhibitors. I have been using 2.5 ug/ml final concentration in PCR
reactions (with avian bone extract as a template). You can use higher
concentrations, but at some level the BSA itself seems to inhibit the
PCR. Another way I have gotten around inhibition problems is to dilute
the template much more than "normal". You may reach the level at which
inhibition is no longer a problem while (hopefully) having some template
left in the reaction.
Cheers,
Tom Quinn
Dept. of Biological Sciences
University of Denver
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