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Denaturing gel electrophoresis for qPCR


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3 replies to this topic

#1 hianghao

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Posted 10 February 2011 - 05:17 PM

I wanna check my RNA for qPCR. I did the absorbance 260/280, and i am going to run a gel for my RNA. Is it a must to run denaturing gel or not?
My lab's recipe for denaturing agarose gel is:
EDTA 1.45g
Boric acid 6.85g
Water 250ml
Final pH 8.3; final conc=20x

According to the recipe, 1x of this buffer is used as running buffer; to prepare the gel, formaldehyde is added to 1x buffer together with agarose. RNA is loaded to the well with normal dye.
However, i can't find any reference for this method and no one knows where does it comes from. Can anyone verify this method for me?

When i tried to google for denaturing gel, most of the articles i found use MOPS and there is an article using normal TBE buffer (loaded with formamide in well). I dont have MOPS and formamide in my lab. Can i use the method with TBE + formaldehyde? IS there any other choices of recipes?

Edited by hianghao, 10 February 2011 - 05:27 PM.


#2 hianghao

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Posted 14 February 2011 - 03:24 PM

Did i post this on the wrong section??

#3 Maddie

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Posted 17 February 2011 - 12:40 PM

Did i post this on the wrong section??



No, you didn't. It just looks like nobody here has extended knowledge on your denaturing gel technique. I sure don't, sorry.
But I still wanted to let you know that we do NOT ignore you :P
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#4 phage434

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Posted 17 February 2011 - 05:45 PM

You can get a good idea of the quality and length of RNA samples with an ordinary gel. Denaturing is required for good length determination, since RNA is generally single stranded and forms secondary structure, but it is easy to see 16s and 23s rRNA bands and smears of mRNA on an ordinary gel. You still need to be careful of RBAse contamination of the gel box, agarose, running buffer and loading buffer, however.




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