Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protocols and Methods Forum
Register Search Today's Posts Mark Forums Read

Protocols and Methods Forum Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


long mouse DNA template PCR, western blot with mouse tail

long mouse DNA template PCR, western blot with mouse tail - Protocols and Methods Forum

long mouse DNA template PCR, western blot with mouse tail - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 10-14-2003, 02:32 PM
Ripley
Guest
 
Posts: n/a
Default 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!
Reply With Quote
  #2  
Old 10-14-2003, 04:57 PM
Peter Ashby
Guest
 
Posts: n/a
Default long mouse DNA template PCR, western blot with mouse tail

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.
Reverse the Spam and remove to email me.
Reply With Quote
  #3  
Old 10-14-2003, 04:59 PM
Peter Ashby
Guest
 
Posts: n/a
Default long mouse DNA template PCR, western blot with mouse tail

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.
Reply With Quote
  #4  
Old 10-16-2003, 09:52 AM
Ripley
Guest
 
Posts: n/a
Default long mouse DNA template PCR, western blot with mouse tail

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>...
Reply With Quote
  #5  
Old 10-17-2003, 11:23 AM
Peter Ashby
Guest
 
Posts: n/a
Default long mouse DNA template PCR, western blot with mouse tail

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.
Reply With Quote
Reply

Tags
blot , dna , long , mouse , pcr , tail , template , western


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
p53 antibody for western blot of mouse tissue !! OK Protocols and Methods Forum 4 11-06-2010 05:35 AM
dot blot and western blot Liang Western Blot Forum 1 08-07-2009 03:26 PM
Production of Monoclonal Antibodies - Technique in Mouse Cellbiogal Antibody Forum 2 01-08-2009 10:52 PM
Mouse Brain Western Blot Detection Denise Western Blot Forum 1 09-17-2008 02:52 AM


All times are GMT. The time now is 08:16 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.14024 seconds with 16 queries