missing 500 bp band w/ lambda hind3

Dr R. Dalgleish ray at leicester.ac.uk
Thu Jul 7 10:39:54 EST 1994


> 
> On Thu, 7 Jul 1994 13:03:27 GMT,
>   BRIAN PETER MERRY writes:
> 
> >Hi,
> >We need some help.  We're running 1% and 2% gels in our lab using Lambda hin3 
> >as a molecular weight marker.  We load 500 ng of lambda in our marker lane and 
> >we don't get resolution of the 500 bp band when prestaining the gel with etbr.
> >Do we need more DNA in the marker lane?  More etbr (we now use 2ul/50ml of 
> >gel, this used to be enough).  Any comments/suggestions will be appreciated.
> 
> If my memory serves me correctly, the 564 base fragment and the 23130 base 
> fragment contain the 2 cos sites/sticky ends of the phage DNA and anneal 
> together. Heat marker to 65C for 5 mins and cool rapidly on ice before 
> loading to dissassociate the 2 fragments.
> DAJ
> 
> David A. Johnston
> Dept of Zoology, The Natural History Museum, Cromwell Road,
> South Kensington, London SW7 5DB. England
> (tel 071 9389297, fax 071 9388754, email daj at nhm.ac.uk)
> 
> 
I'm afraid that your memory fails you. It's the 4361 base fragment that
"sticks" to the 23130 fragment because of the cos ends. The problem is 
really one of the amount of DNA that is available to be seen. In 500 ng
of lambda/HindIII the 564 fragment constitutes about 5 ng. If a marker
is really needed down there, then about 100-200 ng of phiX174RF/HaeIII
should do the trick.

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  Dr Raymond Dalgleish,          JANET: ray at uk.ac.le
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