RNA gel

[Aaina]

Hi guys,

i m continuousely facing a problem while runnig RNA gel. Problem is that the bands run in opposite direction. I mean when i run the gel both dyes (xylene and bromophenol blue) run along the gel but when i view the gel under UV the bands are in opposite direction (almost at the end of -ve termini and are about to go into buffer). If i run it for longer time they goes out of the gel into the buffer. ( i checked my termini they are always at right position)

I repeated the procedure for several times with fershly isolated RNA. Used DEPC H2O wherever required. used 1.2% agarose and for some times 1.5%

plz tell me is it possible that these orange golden bands in the opposite direction are either RNA or it is only EtBr that is shining.

Another problem that is facing by a friend of mine is :her RNA bands are not resolved into 28s and 18s. Moreover she is using DNA ladder as markers and marker bands are also not resolving (she has no option other than using DNA marker).

[danfive]

Sometimes if you load too much RNA it won't travel out of the well, check your concentrations.

Check the pH of your DEPC-treated water, low pH will increase RNA degradation, and DEPC treatment can generate carbonate products that can lower resistivity and hinder electrophoresis. Also check your TBE or TAE buffers.

As far as RNA ladders go I've heard of Fermentas' RiboRuler Rna ladder, but I've never used it.

As last resort you can try PAGE

[Aaina]

thanks for replying

Its not like that RNA does not comes out of the well it does but in oppsosite direction. If i run the gel deliberately in opposite direction dyes go into the opposite direction while rna goes into the right direction its a very strange thing bcoz both the dye and rna shud run in the same direction but it does not

[danfive]

Originally Posted by Aaina

thanks for replying

Its not like that RNA does not comes out of the well it does but in oppsosite direction. If i run the gel deliberately in opposite direction dyes go into the opposite direction while rna goes into the right direction its a very strange thing bcoz both the dye and rna shud run in the same direction but it does not

Some Dyes are dissolved in acidic solutions, or non-treated water, where bromophenol blue would be slightly acidic, and carry a positive charge, therefore migrating to the negative terminal, the opposite of negative charged RNA.

[MARGARITA MARTINEZ]

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Please Some One Will Explain Me.

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Nice Weekend

Margarita

[MARGARITA MARTINEZ]

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Is In Jpg.

Thank You

[aquaplasmid]

Relax. No, that's not RNA. It's free EtBr. You must have added EtBr in your loading buffer, did you? EtBr runs to the negative while your RNA to the positive.