long mouse DNA template PCR, western blot with mouse tail

Hi all!
I am trying to genotype knock-out mice with PCR. I extracted DNA from
tail using standard protocols and I designed a set of primers but all
I get from the PCR is either unspecific or none. I unsuccesfully tried
to optimise conditions. The polymerase I used was Taq Gibco, Phusion
Finnzymes and Expand Long Template Roche. The length of the product is
unknown but the maximum of 14 kb. Has anyone got any experience with
long PCR with mouse tail DNA template? The second question concerns
western blot with proteins obtained from mouse tail. Has anyone done
it and can give some advice?
Thanks for any suggestions!

In article <[Only registered users see links. ] >,
[Only registered users see links. ] (Ripley) wrote:


You'er trying a genotype with a 14kb pcr? give it up and redesign on
something that will be much more robust or do a Southern. Take it from
me, you absolutely, completely and utterly DO NOT WANT an unreliable
genotype method. It will take over your life, mess up your mouse colony
and make you thoroughly miserable. Genotype methods need to be as robust
as you can make them: a 300bp vs a 500bp band for eg. 14kb just does not
meet that criterion.

Peter

--
Peter Ashby
School of Life Sciences, University of Dundee, Scotland
To assume that I speak for the University of Dundee is to be deluded.
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In article <[Only registered users see links. ] >,
[Only registered users see links. ] (Ripley) wrote:


Oh and if you want somewhere to ask these questions from been there,
done that people, subscribe to the transgenic mouse list. More info at
<http://mailman.ic.ac.uk/mailman/listinfo/transgenic-list>

Peter

--
Peter Ashby
School of Life Sciences, University of Dundee, Scotland
To assume that I speak for the University of Dundee is to be deluded.
Reverse the Spam and remove to email me.

Thanks for the advice. I am also trying with shorter PCR, good to know
others also do not find the long one so easy. As for the southern, the
quality of the template will not be a problem?

Iwona

Iwona Grad
[Only registered users see links. ]
University of Geneva
Departement de Biologie Cellulaire

Peter Ashby <[Only registered users see links. ].uk> wrote in message news:<[Only registered users see links. ].uk>...

In article <[Only registered users see links. ] >,
[Only registered users see links. ] (Ripley) wrote:


I've not done too many tail southerns but I do my pcrs on raw digest
after heat killing the PK, the protocols for southerns I have spool the
precipated dna to get both good purity and intact dna.


Say hi to my sometime collaborators Denis Duboule and Joska Zakany if
you bump into them ;-) In fact go over to zoology and chat to the people
in Denis's lab (3rd floor iirc), they mostly use southerns I think but
do some pcr too.

Peter

--
Peter Ashby
School of Life Sciences, University of Dundee, Scotland
To assume that I speak for the University of Dundee is to be deluded.
Reverse the Spam and remove to email me.