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RNA electrophoresis

electrophoresis RNA troubleshoot

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#1 carola26_23

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Posted 16 December 2011 - 03:52 PM

Hi everyone, I have a question about RNA electrophoresis. During the electrophoresis from my RNA I saw only one very sharp band in the gel, contrary to the 18S and 28S bands that I usually get. I am very sure that my RNA is not degraded, bacause I´ve being using the same samples before and they always look great. The conditions for the electrophoresis were: agarose 1%, TAE 1x buffer, 5 v/cm during 40 min.

I will be really gratefull if someone can help me.

Sorry about my english.


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#2 bob1

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Posted 19 December 2011 - 11:44 AM

From the picture it is a little hard to tell what is going on - your ladder hasn't separated well at all, so I can't estimate the size of the bands you have.

Sometimes you will only see one of the rRNA bands when you do an extraction, this is usually because you have a low concentration extraction. It could also be that you have migrated the lower bands into the ethidium shadow. You can overcome this by post-staining your gel.

#3 carola26_23

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Posted 20 December 2011 - 05:37 AM

I did not uploaded a pictuce with the ladder more separated because at that point I can´t see the RNA, but the RNA it is always below the ladder, wich indicates that the product I am seeing it is lower than 100 bp. At this point I am almost sure that my RNA is degradated in a very weird way. :P

#4 bob1

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Posted 20 December 2011 - 11:41 AM

It is pretty odd degradation to all be restricted to one fairly tight band, normally you would expect to have a smear of some sort. If the band is, as you say, less than 100 bp, then it is unlikely to be useable. Perhaps you have some RNase contamination somewhere in your process.

Anyway, it seems that you will have to repeat the experiment and extract more RNA.

Edited by bob1, 20 December 2011 - 11:41 AM.


#5 WSN

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Posted 06 January 2012 - 09:29 AM

To run RNA in plain agarose gel, you may want to treat your RNA with glyoxal loading buffer (lonzabio.com) and heat at 65C for 15 min. Otherwise you are relying on you luck to see expected bandings. Good luck!





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