Question about Formaldehyde gels and Northern Blots

bradley turner turner at sprcore.caregroup.harvard.edu
Fri Mar 10 14:33:20 EST 2000


Hello,

I've always used a downward alkaline protocol (see references
below) with no problem.  I've even been able to strip and
reprobe blots ~5 times.

Hope this helps,
Brad Turner


****************************************************************
                    Bradley Turner
                Beth Israel Deaconess
                    Medical Center

Harvard Medical School          617-667-1215 phone
Division of Gastroenterology    617-667-2767 fax
Room Dana 536                   bsturner at biosun.harvard.edu
330 Brookline Avenue            bturner at caregroup.harvard.edu
Boston, MA 02215                turner at sprcore.caregroup.harvard.edu
****************************************************************



On 1 Mar 2000, 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
> 
> ---
> 
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downward alkaline northern references from PubMed



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