Question about Formaldehyde gels and Northern Blots

John Thompson jrt at home.com
Sat Apr 1 19:51:48 EST 2000


>3) Could the RNA be degrading in the Formaldehyde gel?  

If you haven't stained, how do you know the RNA was intact to begin
with? I doubt that it is degrading during the run.  I routinely add
EtBr to my RNA samples and photograph the gel before blotting (working
from memory I add 1 ul of 50 ug/ml EtBr to each sample).  Adding it to
the sample gives less background staining, thus more contrast in the
picture.  Like others, I only wash briefly in 10X ssc (2X, 30 minutes
total) before setting up the transfer. I haven't done a rigorous side
by side with/without EtBr staining; but it clearly doesn't cause a
dramatic reduction in signal.

If you are just using the RNA for hybridizations, try dissolving the
RNA in 0.02% DEPC. This will inactivate any potential RNAse that may
have carried through with the prep.  Ambion has some newer reagents
along these lines that I have not tried yet but which are claimed to
inhibit RNAses and be compatible with enzmes that might be used later.

Regards,
John Thompson
Merck Research Laboratories 


smitam01 at holmes.ipfw.edu (Alan Smith) wrote:

> Hello,
>
>I have some basic questions about capillary transfer of RNA from
>formaldehyde gels to nylon membranes.  We have been following the
>protocols for RNA gels and RNA transfers from the second edition of
>Maniatas.  I do not believe the problem is with the isolated RNA because
>I have checked it multiple times on an agarose gel and it seems to be
>intact.  Some basic info about what we are doing.....We are trying to
>probe for an abuntant 2.5kb transcript in Arabidopsis using a full
>length cDNA as a probe.  The probe is labeled by random priming using
>32P.  We are pehybridizing and hybridizing in 50% formamide, 6X SSPE, 5X
>Denharts, 1% SDS, and 200ug sheared herring sperm DNA.  We prehybe and
>hybe overnight.
>The questions I have are
>1)  Is the formaldehyde blocking transfer?  We wash the gel 3 times at
>20 min each in DEPC treated water and then soak it for 45 minutes in 10X
>SSC.
>2) Are we using the correct transfer buffer for Nylon membranes?  We are
>doing a transfer overnigth with 10X SSC.
>3) Could the RNA be degrading in the Formaldehyde gel?  We do not stain
>the gel before transfer because EtBr will block the transfer of RNA.
>Should we stain the gel before transfer?  We load equal amounts of RNA
>based on gel and Spec readings.  We are also going to strip and reprobe
>the membranes with the house keeping gene CAB as a control.
>4)  Are there more ideal ways to conduct a northern transfer from
>formaldehyde gels other than what is written in Maniatas?
>
>Thank You
>
>Alan Smith
>graduate student
>department of biology
>IPFW
>email: smitam01 at holmes.ipfw.edu
>
>---





More information about the Methods mailing list