Question about Formaldehyde gels and Northern Blots
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.
Merck Research Laboratories
smitam01 at holmes.ipfw.edu (Alan Smith) wrote:
>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
>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
>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?
>department of biology
>email: smitam01 at holmes.ipfw.edu
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