Multiple bands on RNA gel, why? - (Apr/13/2006 )

Hi,

I was told that a RNA sample will give 3 bands in an agarose gel, i.e. each showing 28S, 18S and 5S.

However, could anyone pls enlighten me on my gel picture which shows multiple bands for most lanes? Extraction method was via Trizol.

Thank you.

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well. As your marker is not very good (regarding migration i mean) i think the main problem comes from the gel. I think an other one is more suitable before further experiments.
Maybe it was not enough solidified.

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or was melting during the run.

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was using a 1.5% agarose gel in 1xTBE. ran on 90V for approxly 1hr. during the run, the gel tank was surrounded with ice.

what kind of gel do you suggest i use, fred_33?

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hi
How do you prepare your gel first?
suggestion : melt agarose in water carefully, and let it cool in waterbath to 55°. Mix formaldehyde and formamide and put them no more than 5' in the waterbath. After these 5' mix with the agarose melted, mix and pour immediately under hood. Let it cool for 1h approx. I mean this step was maybe the critical one. moreover formamide and formaldehyde doesn't like high temperatures.

second : voltage is ok i think. there is guidelines in maniatis for a certain voltage per centimeter. i run it in 1X MOPS.

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also, formamide tends to decompose when mixed with water so use the gel as soon as possible.

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hello guys,

thanks a lot for the pointers. will take a shot at your suggestions, hopefully will get a better run.
appreciate both your quick inputs to my 2 problems lately

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