formaldehyde gel for RNA
przemko at sgi.celgen.kuleuven.ac.be
Tue Jun 18 02:07:16 EST 1996
> I'd tried to run my RNA sample on formaldehyde agarose as protocol on
> Molecular cloning by Sambrook several times, but I get smear bands for
> samples and marker and failed to get two distinct band for 28S and 18S as
> expected. I'd eliminated the possibility of RNase contamination. Is these
> caused by quality of formaldehyde or pH? Can I have references for these
> Thanks in advance.
I do these gels all the time. I must tell you that most probably your
RNA is degraded. Other possibilities include
- gel overloading (very common- you should put NOT more than 10ug total
per lane in BRL Horizon 11x14 and a 10 well comb)
- too fast run (MOPS should not be abused- 100V in the above setup is
- if you run the gel long time you should recirculate the buffer
- your formamide for the denaturation should be DEIONIZED. Try to freez
small aliquots and then use each of them two- three times.
- to be sure the quality of your RNA you can run a normal TBE gel (just
make sure that all is RNase free. TBE has to be made from a DEPC treated
water. You cannot treat TRIS with that stuff). On that gel what you
expect is a lot of smear and two VERY strong ribosomal bands. If you
don't see that then your RNA is degraded.
Hope that this helps
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