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Differential Display

Rover rover at ns1.livnet.com
Fri Dec 1 20:50:27 EST 1995


smori at nmsu.edu (Shahram Mori) wrote:

>@ wrote:
>: I have been doing differential display reactions and get good gels and can
>: even amplify the bands out of the gel , But when I do northerns with these
>: reamplified bands there isn't any signal at all on the blots. I know my
>: RNA is fine and the transfer is fine too. Can't figure out what the
>: problem is. 
>: Any help would be really appreciated.
>: Thanks,
>: Aardraa Potnis
>: apotnis at aim.salk.edu

>This is what's been ruining my life for a year and a half. I get the 
>exact same thing. *they* say that Northerns blots are not a good thing.
>Some people say to clone and sequence first (because you might be looking 
>at a lot more than one sequence) and then do northerns.
>Others say do RPA's first....... BUT I don't see why northerns don't
>work.

>p.s. I will bet that this method is MAINLY luck and has nothing to do with
>consistent protocols.

Grin... I don't subsrcibe to the "luck" philosophy unless your
isolating clones from a library and get it on you first round. That is
luck.

 	The reason Northerns may not work is your gene in question may
be detectable using PCR, which is much more sensitive , but expressed
at low levels undectectable by Northern. Ask anybody who has had PCR
contaminations and they will gladly tell you how sensitive the
technique is.

 If you ar still interested in genes that may have this low level of
expression try the Northern first, followed by sequencing , RPA , PCR
or whatever. Northerns are the fastest technique in our hands so we do
it first, and with the use of nylon or other repeated use membranes we
can generate a lot of hybridizations without using buckets of RNA.






--Rover The Mad Molecular Biologist
rover at livnet.com
barlow at picard.evms.edu




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