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RNA agarose gel electrophoresis came up empty - (Jul/08/2014 )

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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! 

-Thyago Leal-

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?

-bob1-

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

-mdfenko-

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. 

-Thyago Leal-

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. 

-Thyago Leal-

 

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.

-mdfenko-

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

-mlomonaco-

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

-mlomonaco-

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

-merlav-

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.  

-Ahrenhase-
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