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I am having troubles running RNA on a gel.
Do I need to run the RNA with formaldehyde or formamide?
Do I need to heat the RNA to melt secondary structure?
I am using 1 % agarose gel for RNA.
Re: RNA gels
I understood that you need to have completely denatured RNA. Then you should mix the sample with good quality loading dye solution that contains formamide (like Fermentas 2X RNA Loading Dye Solution (#R0641)), heat at 70ºC for 10min, chill on ice for 3 minutes and load onto the gel.
Best would be to use denaturing formaldehyde agarose gel in MOPS buffer according to the below protocol:
• Freshly prepare 10X MOPS buffer:
0.4M MOPS (pH 7.0),
0.1M Sodium acetate,
0.01M EDTA (pH 8.0).
• Prepare 1% gel as follows:
-Stir 1g of agarose powder in 72ml of deionized water
-Melt the agarose, and then add 10ml of 10X MOPS buffer and mix.
-When the agarose solution cools to 60°C, add 18ml of fresh formaldehyde (37%) in a fume hood and mix thoroughly.
- Pour the gel.
• Place the gel into an electrophoresis apparatus containing 1x MOPS buffer
• Heat the RNA samples and ladder at 70°C for 10min, and then chill on ice for 3 minutes. Load onto the gel.
Try, I believe this will work
Re: RNA gels
I haven't found success in using the above protocol. The bands either show up extremely faint, or they never show up at all.
I am thinking that this could be an RNase contamination problem not directly associated with the gel. I found a kit online called the Elementary RNA Gel Kit made by amresco that simplifies the whole process. You don't have to add formaldehyde, formide, or ethidium bromide. I am trying that out right now to see if the results are better.
Here is the link if you are interested: amresco-inc.com/catalog/PDF/Q1%202006%20INTL.pdf
Re: RNA gels
How was the Elementary RNA gel kit? I'm considering to try it out soon, but wondering about which kit I should select.. There are a few on the market.
I'm going to use it to analyze an in vitro synthesis.
Does the kit contain everything needed to perform the electrophoresis, or do I have to buy any additional reagents?
Would greatly appreciate an answer
|gels , rna|
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