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electrophoresis for RNA - agarose gel or FA gel?? (Oct/21/2007 )

sorry...
i still in hesitate which will be more suitable to run electrophoresis for my RNA...
whether better with agarose gel or FA gel...

its because agarose gel enable me to discover if theres any DNA contamination...
but FA gel gives me better view of RNA... however, FA gel cant detect any DNA contamination, right??
look forward for ur answer...
thanx...

-parami-

agarose gel.

it's cheap, easy, and you can see nice clean bands.

i'm nice today, so here is a protocol:

[attachment=3736:AgaroseR...aldehyde.pdf]

V

-vetticus3-

i follow the same protocol but its great to see the whole protocol in a pdf file. thanx vetticus

-T. reesei-

Excuse me my ignorance, what is FA gel?

-aztecan princess-

formaldehyde agarose gel

-mdfenko-

QUOTE (mdfenko @ Oct 23 2007, 07:37 AM)
formaldehyde agarose gel


We run RNA in a normal agarose gel for routine, when we want a good picture (for a report, a paper or something) we do Formaldehide-agarose gel.

-aztecan princess-

things is that formaldehyde denaturates the RNA and not standard agarose gel.
That's why it's for better pictures
but formaldehyde is a poison so it's always nice to reduce toxic use.

by the way, thanks v for the protocol. but i have a question : what's the role of the ammonium acetate in solution ? I meantoes it allows to fix RNAs ?
And a point : you may add 0.4┬ÁL of a 1mg/ml BET solution to your samples. You'll then don't need to stain the gel and can take a picture quicker. (obviously this point does not apply if ammonium acetate fix RNAs).

-fred_33-

thanx for the protocol...
btw, i discover a weird things on my gel...
in fact, for RNA works, the gel shld only have the 18S and 28S ribosomal RNA bands and some lower molecular weight smear...
but for my stuff, i got a band above the 28S RNA band, around 1cm above from the gel picture...

at first, we thought it might be DNA, but after run with PCR, it doesnt show any product..
and when RNase A to treat it, the band disappear also...

so, we now in the doubt whether it is also the RNA band...

do u all ever face this kind of problem??
thanx for help...

-parami-

QUOTE (fred_33 @ Oct 28 2007, 09:04 PM)
by the way, thanks v for the protocol. but i have a question : what's the role of the ammonium acetate in solution ? I meantoes it allows to fix RNAs ?


if you have insufficient staining of denatured RNA, adding ammonium acetate with ethidium bromide helps.
i think it has something to do with solubility. i dont' know. it works. i'm happy.

parami... never had that problem. sounds like contamination. how do you extract the RNA?

V

-vetticus3-

[/quote]

if you have insufficient staining of denatured RNA, adding ammonium acetate with ethidium bromide helps.
i think it has something to do with solubility. i dont' know. it works. i'm happy.

parami... never had that problem. sounds like contamination. how do you extract the RNA?

V
[/quote]

i use the mortar and pestle make the cell become powder, then use trizol reagent in rna extraction...
every time extraction, got a 'alien' band appear...

things is ok when they use the dismembrantor to make the cell become powder form... no contamination and strange things happen...

by the way, will it be better to manual protocol in rna extraction...
coz i need to use the rna for later cDNA library construction works...

thanx alot...

-parami-