Question about Formaldehyde gels and Northern Blots
smitam01 at holmes.ipfw.edu
Wed Mar 1 19:40:29 EST 2000
Addition to my earlier submission
We are not getting any hot bands just a large or small amount of background
depending on washing conditions . We have tried three different probes which
were denatured by boiling and then put on ice before addition. We have
hybridized other membranes (Southerns) at the same time as the northerns and
recovered good hybridization on the southerns, but not the northerns. I think
our problem must be in the transfer from Formaldehyde gel.
Thank you for all of the excellent advice so far
"Hiranya S. Roychowdhury" wrote:
> At 07:29 PM 3/1/00 -0000, 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
> What is the "problem"?
> >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
> Formaldehyde does not inhibit transfer since it does not stay in the gel for
> long after the transfer is initiated. There is no need to wash the gel in
> DEPC treated water (actually, it is best to avoid it altogether).
> You have not actually mentioned anything about the "problem" itself. Are you
> not detecting any signal in the autoradiogram? If so, then it is possible
> that, other than degraded RNA, you are leaching most of the RNA away during
> the prolonged wash-soak steps.
> Eliminate the 3x20min water wash. Rinse the gel briefly with autoclaved or
> MilliQ grade water and equilibrate it in the 2-5x SSC or SSPE for 10min with
> gentle shaking. Set up the transfer with 5x SSC or SSPE if you are doing it
> >2) Are we using the correct transfer buffer for Nylon membranes? We are
> >doing a transfer overnigth with 10X SSC.
> see above; with 10x SSC the transfer shoud be complete in 3-4 h.
> >3) Could the RNA be degrading in the Formaldehyde gel?
> That is entirely possible.
> >We do not stain
> >the gel before transfer because EtBr will block the transfer of RNA.
> That is absolutely not true. EtBr does not, in anyway, block NA transfers.
> Formaldehyde gels do not usually contain EtBr since it the RNA pick up EtBr
> as well from the formaldehyde gels. I know that the manual says EtBr
> inhibits transfer, but I have not found it to be so. EtBr, in the entire
> membrane, however does cause problem during hybridization.
> To visualize the RNA, you should include EtBr in the RNA sample buffer,
> either before or after denaturing the samples. Then, you will be able to
> actually see if the trasfer has worked.
> >Should we stain the gel before transfer?
> no, see above.
> 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?
> The membranes used these days are extremely efficient. Transfer of NA's will
> occur almost under any condition. The high salt keeps the capilary transfer
> from diffusing and acts to carry the NA's faster.
> If you are using HybondNX or N+ from Amersham, they also recommend alkaline
> transfer (yes... even for RNA) using 0.5M NaOH. People have had good results
> with it. I have not tried it myself since the SSC/SSPE capillary transfer
> works just fine for me.
> Finally, I would switch to Church & Gilbert's buffer system for NA
> hybridizations if I were you.
> >Thank You
> >Alan Smith
> >graduate student
> >department of biology
> >email: smitam01 at holmes.ipfw.edu
> Dr. Hiranya Sankar Roychowdhury
> GENE LAB/ EPPWS
> New Mexico State University
> Las Cruces, NM 88003
> Ph. (505) 646-5785
> hroychow at nmsu.edu
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