missing 500 bp band w/ lambda hind3 (fwd)

IZZY7KI at MVS.OAC.UCLA.EDU IZZY7KI at MVS.OAC.UCLA.EDU
Sat Jul 16 18:10:00 EST 1994


In article <3044km$ss2 at news.cs.tulane.edu>,
schneid at rs6.tcs.tulane.edu (Jason Schneider) writes:

>Robert Preston (rapr at MED.PITT.EDU) wrote:
>: >
>: > 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.
>: >
>Are you heating the marker before you load it? Lambda has cos ends that really
>like to link up. If one of the bands on your lane is unusually bright, that
>500 bp fragment may mixed up in there. Heating at 65^C for 10 minutes should
>do the trick. If that is indeed your problem.
>Only IMHO
>--
>Jason S. Schneider
>Tulane University
>-------------------------------
>schneid at mailhost.tcs.tulane.edu
>
>"With imagination, I'll get there..."
>          --Harry Connick, Jr.

I think you should try loading more lambda DNA.  You currently load
500 ng of DNA, of total size 48kb (add up the bands).  Since the
bands are of equimolar concentration, the 500 bp band is present at
about 500ng x (500bp / 48,000bp) = 5 ng.  I would not expect to see
this amount of DNA on an ethidium-stained gel.
     By the way, it is true that two bands tend to associate by
virture of overhanging ends.  Theses are the 4kb and 23kb bands
(on a HindIII map of lambda you will see these are the end fragments)
and they form a 27kb band.  Under the right gel conditions you will
see this.  Heat at 65 deg. for 15-30 seconds is enough to dissociate
the 12-bp complementary region and restore the 4kb band.

Best of luck,

Benjamin S. Braun, UCLA



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