Problem with TriReagnt - (Mar/03/2007 )

i have made a trizole reagent using following receipe


{21ml of AGPC (TRI Reagent)
- Mix 10 ml 4M guanidinium thiocyanate, 352ul 0.75M sodium citrate (pH 7.0) and 528ul 10%
sarcosyl
- Add 76 μl 14.3 M ß-mercaptoethanol -Sigma- [total final volume=10.9 ml].
- Place 10 ml of this mixture in a new tube, add 1 ml 2M sodium acetate (pH 4.0) and 10 ml watersaturated
phenol. This is the final AGPC solution (This solution is good for at least 2 months at 4oC).}


i had some trizole reagent ,which i used for rna isolation but finished.and i prepared new one using that receipe but problem i am facing is that rna yield is so much reduced as i isolate before.
if any one have plz any suggestion to increase my yield.because rna yiled is so small that i cant solubilize it in more than 10 ul of depc water and than whole mixture used in one pcr(250 ul)

therefor i need some way out of this problem
plz help me

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Where did this recipe come from? The "water saturated phenol" sounds as if it is ambiguous. The pH of this solution is unspecified, but clearly important to the final pH of the mixture. Phenol is very often equilibrated with Tris-HCl at pH 4.5 (for RNA work) or pH 7.5 (for DNA work). Or they could mean unbuffered phenol. It probably matters a good deal what the initial pH of the phenol you start with is.

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I agree. The final pH of the solution is in doubt. Moreover this recipe relies on the phenol to acidify it to pH 4.5. The remainder of the solution is probably quite close to neutral due to the buffering capacity of the trisodium citrate.

I might also add that the molarity of sodium acetate is rather low. 0.1M. I would bring it up to at least 0.3M, preferable to 0.4M. Perhaps that is another reason why recovery is poor.... the RNA isn't percipitating.

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i can not make new trireagent ,because my supervisor will not allow me.

is there any space for modification of this solution,to improve yield of rna.

i have used distilled phenol.and i am preserving trireagent in fridge.

thanx


i got that receipe from following site
www.flemingtonlab.com/Protocols/TotalRNAprep.pdf

and i have also tried to attach.

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Well, the link clears things up -- they mean unbuffered phenol. The only potential problem I see might be the pipetting of the phenol -- I presume you know that the phenol will be the bottom layer of the water saturated mixture. If you use the top water layer, things will definitely not work.

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i have used distilled phenole and it was not buffered.




thank u sir

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you should still try to get a pH, I think the pH of the top layer should reflect the pH of the phenol?? Most likely you have a pH problem... as phage434 said, you want 4.5 for RNA work... there is something about phenol oxidizing and ?changing pH....

Good Luck

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usually the top layer is the product by which the phenol is saturated.
Mean TE for DNA and citrate (pH 4.5 - 4.7) for RNA
so the pH of the upper phase is quite an illustration for the pH of bottom (phenolic) phase.

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can i make (change) ph of my trireagent up to ph 4.5

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i think yes. Just remove as you can of the upper phase and add citrate til saturation
for checking the pH, found that ref...

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