Denaturing Agarose Gel Protocol (Inside)

[John Buckels]

Due to popular request I have decided to post my protocol for Formaldehyde Denaturing AGE

1.2% FA Gel Preparation

NB: the gel must be cast in the fume hood
To prepare FA gel (1.2% agarose), mix the following in a specified conical flask:
·1.2 g agarose
·10 ml 10x FA gel buffer (see composition below)
·Add RNase-free water to 100 ml

Heat the mixture to melt agarose. Cool to a holdable temperature. Add 1.44 ml of 37% formaldehyde and either 3 µl of SYBRgreen II stock solution or if preferred, Ethidium Bromide to a final concentration of ~200 ng/ml. Mix thoroughly and pour onto the specified gel support in the fume hood. Insert the specified combs. Prior to running the gel, equilibrate in 1x FA gel running buffer (see composition below) for at least 30 min.

RNA sample preparation for FA gel electrophoresis

Add 1 volume of 5x loading buffer (see composition below) per 4 volumes of RNA sample (for example 2.5 µl of loading buffer and 10 µl of RNA) and mix.
Incubate for 10 min at 65°C in the thermal cycler, chill on ice, and load onto the equilibrated FA gel.
Run gel at 50–70 V/cm in 1x FA gel running buffer.
If Ethidium Bromide is used the gel may require de-staining in 1x FA Gel Running Buffer for 10 minutes to reduce background

Composition of FA gel buffers

10x FA Gel buffer
·200 mM 3-[N-morpholino]propanesulfonic acid (MOPS) (free acid)
·50 mM sodium acetate
·10 mM EDTA
·pH to 7.0 with NaOH
·Toxic. Take appropriate safety measures.

1x FA Gel Running Buffer
·100 ml 10x FA gel buffer
·20 ml 37% formaldehyde
·RNase-free water to 1 litre

5x RNA Loading Buffer
·8 µl saturated aqueous Orange G solution
·40 µl 0.5 M EDTA, pH 8.0
·360 µl 37% formaldehyde
·1 ml 100% glycerol
·1542 µl formamide
·2 ml 10 x FA Gel Buffer
·RNase-free water to 5 ml

[seemabshaikh]

Hi,
Can you please describe the role of each reagent in this ?
I mean like Formamide disrupts base-pairing and so helps in denaturing the RNA (second structure removal). I guess formaldehyde has the same purpose. But if so do u know why both are used while doing denaturing gel electrophoresis?
Also what is the role of MOPS ?
Thanks
Cmab

[phage434]

Did you really mean 2ml of 10x FA gel running buffer in your recipe for 5x RNA loading buffer? Given that you haven't got a recipe for 10x FA gel running buffer, and that you're already adding formaldehyde to the loading buffer, I suspect that you really meant 2 ml of 10x FA gel buffer in the loading buffer recipe.

[John Buckels]

You are absolutely right, i've edited the protocol so it is accurate

[AGMN]

Hi John

First of all, I have to thank you for editing this protocol. And may I ask that do you think it is better to add the EtBr onto the gel or with the loading buffer?

Thanks

[John Buckels]

This has been debated many times on thes boards. I always add my stain directly to the gel to ensure uniformity, but I think the general concensus is that you can do it whichever way best suits you

[stevenzhong]

very useful protocol. we use a similar one with Na-PO4 buffer. Your 5x sample buffer recipe is better then our 2x.

we adjust the ph of the formaldehyde to 7 by adding NaOH.

I wonder if the MOPS can tolarate the acidic formaldehyde. We can use it directly and skip the ph adjusting step if it is strong enough.

[starter]

Although its a new forum, you can still find lots of general biology protocols, DNA and RNA techniques on http://www.fpilus.com/forums/ that you may find helpful regarding this topic

[David Spencer]

To respond to several questions:
-The formaldehyde reacts chemically with the amines of particularly G's (but also C's and I assume A's) and therefore prevents base-pairing (and hence hairpins). The formaldehyde adduct is only fairly stable at or below a pH of about 7, hence the MOPS buffer (pKa of 7.15). There was an attempt to replace formaldehyde with glyoxal (which is two formaldehydes 'back-to-back') but that never seems to have caught on (it is less convenient to work with than formaldehyde). Note that formamide is only in the loading buffer because once the RNA is denatured and has reacted with formaldehyde the formamide is unnecessary.
-Putting the ethidium bromide (at excess) only in the loading buffer works very well and gives minimal background to the gel when viewed in UV.
-If your formaldehyde is very acidic when measured in a pH meter it contains too much formic acid (the oxidation product of formaldehyde) and really shouldn't be used.
-Why on earth is the 10X FA gel buffer designated "toxic"? Norman Good evaluated this issue in a 1980 Anal. Biochem. paper (where he was senior author) and MOPS (like all but one of his Good buffers) was found to be quite compatible with mammalian cells in culture as well as with E. coli and a unicellular chlorophyte; the one cell not happy in the Good buffers (particularly Popso) was a cyanobacterium, and the proposal in that case was the toxicity was caused by a negative effect on photosynthesis.

[rima250]

Thank you very much for your helpful protocol. I want to ask whether I can omit ''8 µl saturated aqueous Orange G solution'' or replace it by something else.
Regards.

[tyranosaraus]

I am trying to run a denaturing agarose gel (formaldehyde) to analyze some RNA samples. A lot of protocols suggest adding EtBr to the sample loading buffer/dye, allowing visualization of the RNA during the gel run. I tried this and the EtBr just ran off the gel toward the anode, floating around in the running buffer, while the RNA ran the other way (as normal). I ended up having to stain the gel by sloshing it around in EtBr/buffer after the run, which is what I'm trying to avoid.

Can anyone tell me what's going on?

[relaxin]

You need to add EtBr in the buffer tank too. I think it is better just to stain the gel after the run. Less toxic waste to deal with later.

[mchlbrmn]

Yes, ethidium Br migrates in the opposite direction as nucleic acid. Might have something to do with why they like each other (although may be more hydrophobic interaction?). I once tried EtBr in DNA loading dye, and it didn't work well.
Relaxin, this is a formaldehyde gel, so the entire thing is toxic waste regardless of EtBr.
When I used to do Northerns commonly, I'd cut out the sizemarker lane after the run, and stain only it, and then photograph it with a ruler. When I blotted the rest of the gel, I marked where the comb was, and cut it off here, and marked with a pen on the final Northern exposures where the size marker bands measure to. I'd hybridize the Northern with a housekeeping gene before or after the other hybridization(s) instead of looking at rRNA for RNA loading. I had read that EtBr inhibits the RNA from transferring to the blot. I don't know how serious that inhibition is. It can't be too bad since people do commonly use EtBr staining.

[toptea]

Hi, I am new to this field, wanted to know if there is any protocol that uses SYBR Gold or any other dye instead of etBr???????
Please help me. Regards

[3kids&aphd]

Has anybody tried running RNA on a bleach gel? It's quick and easy and, for me, worked well to confirm quality and quantity of my RNA samples. It's discussed in this paper: http://www.ncbi.nlm.nih.gov/pubmed/22222980, and seems to denature the RNA partially to completely. My own gels showed lovely rRNA bands that ran a little smaller than expected but were clean and sharp and clearly in the expected pattern relative to each other.