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RNA agarose gel electrophoresis came up empty

RNA EMPTY ELECTROPHORESIS EMPTY LANE LANELESS

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10 replies to this topic

#1 Thyago Leal

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Posted 08 July 2014 - 12:57 PM

Hello everyone,
 
After my RNA Extraction using Trizol Reagent (Life Technologies) I  quantified my RNA (and came up 866,54 ng/ul) and then, ran a agarose gel electrophoresis (0,8%) using 3 ul of that RNA + 2 ul formamide + 2 ul load dye.
 
I saw nothing, while I expect a "smear". 
 
Any help?
 
Oh, and I tested other reagents like: Ethidium bromide, agarose, and everything is  ok.
 
Thank you! 


#2 bob1

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Posted 08 July 2014 - 02:54 PM

What did the spectrophotometer curve look like?  Did you get a 260:280 ratio?  How about 260:230?

 

How long did you stain for?  Did your ladder show up?



#3 mdfenko

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Posted 09 July 2014 - 04:53 AM

what is the condition of your formamide? it decomposes when in contact with water.


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#4 Thyago Leal

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Posted 09 July 2014 - 05:35 AM

What did the spectrophotometer curve look like?  Did you get a 260:280 ratio?  How about 260:230?

 

How long did you stain for?  Did your ladder show up?

For all my four samples, their respective data.

 

Sample 1: 546,56 ng/ul

A260/A280: 2.415

A260/A230: 0,770

 

Sample 2: 118,09 ng/ul 

A260/A280: 2.101

A260/A230: 0,368

 

We add the Ethidium bromide when the gel is cooling but not solid. I haven't included ladder because we are short stock of it, due to holidays and delay in delivery. But everying is working in DNA electrophoresis.

 

Sample 3: 834,89 ng/ul

A260/A280: 2.666

A260/A230: 0,952

 

Sample 4: 317,06 ng/ul

A2360/A280: 2.237

A260/A230: 0,657

 

Thank you. 



#5 Thyago Leal

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Posted 09 July 2014 - 05:36 AM

what is the condition of your formamide? it decomposes when in contact with water.

It is not my prep, should I prepare one new? 

Do you have any tips?

Thank you. 



#6 mdfenko

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Posted 09 July 2014 - 07:15 AM

 

what is the condition of your formamide? it decomposes when in contact with water.

It is not my prep, should I prepare one new? 

Do you have any tips?

Thank you. 

 

you can deionize the formamide. here is the procedure used by beckman-coulter:

 

1) put 100ml formamide into a 250ml erlenmeyer flask. check the pH with pH paper (not electrode).

 

2) add 5gm mixed bed ion exchanger (ag501-x8(D) from bio-rad (catalog# 143-6425) or equivalent). stir with magnetic stir bar. if initial pH of formamide is less than 7 then stir for 45 minutes. if the pH is greater than 7 then stir for 60 minutes. keep the flask covered while stirring.

 

3) check the pH. if the pH is greater than 7 then continue. if the pH is less than 7 then discard it and start again with a fresh bottle of formamide.

 

4) filter through a 0.2um nylon filtration unit (nalgene #153-0020 or 151-5020 or corning #430771 or 430773).

 

5) store in single use aliquots at -20C in a non-frost-free freezer.


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#7 mlomonaco

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Posted 09 July 2014 - 07:38 AM

If your gel box is used to run classical plasmid mini-preps, it is grossly contaminated with RNAse



#8 mlomonaco

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Posted 09 July 2014 - 07:49 AM

Your 260/280 ratios are odd.  Please post a spectrophotogram for the members to see.



#9 merlav

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Posted 09 July 2014 - 12:16 PM

please post the photo of the gel and how you prepare the gel


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I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
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#10 Ahrenhase

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Posted 09 July 2014 - 01:58 PM

For what it's worth, I regularly run non-denaturing RNA gels (w/o formamide/formaladehyde/urea) and see bands.  so even if it were to decompose I'd still expect to see a band.  


Edited by Ahrenhase, 09 July 2014 - 01:58 PM.


#11 merlav

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Posted 14 July 2014 - 04:47 AM

Next time use the MWM it will help not only to know the bp of the sample but also will help to know if the gel is well stained, if you ran it backward (at least once in your life will do it), etc. It gives important information so never be cheapskate with it. 


Science without religion is lame, religion without science is blind.
Albert Einstein

I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
Marie Curie





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