Question about Formaldehyde gels and Northern Blots

Alan Smith smitam01 at holmes.ipfw.edu
Wed Mar 1 14:29:24 EST 2000


 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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