I've prepared my acetosyringone by dissolving 196.2 mg of it in both 10 ml of DMSO and ethanol separately. Acetosyringone prepared in DMSO precipitated out when I added it into my autoclaved MS medium. As for ethanol-dissolved acetosyringone, it evaporated as soon as I added it into my autoclaved medium.
Can anyone suggest why the precipitation occured?
We generally prepare 1ml stocks of acetosyringone dissolved in DMSO and have never found any precipitation problems. May be the media was hot when u added aceto.
Yes, I added it while the medium temperature is still around 50 to 60 degree celsius. That means I should only add aceto into the medium at lower temperature?
Thank you in advance.
I also used to add acetosyringone at room temperature, I have never found any precipitation
I never saw acetosyringone precipitation. Check if the reactive is good, and also check if you are preparing the acetosyringone stock in the right concentration.
I dont understand how can be evaporated the Ac if it is dissolved in ethanol and you add to the liquid media after autoclaving...
Hi,
I use Acetosyringone at 200mM dissolved in Ethanol. But normally I put the powder in 1 to 5 ml of Ethanol 96º and then adjust to my final volume of 10 ml (you did it in 96º ethanol only?). In this way, when you freeze it, it can precipitate but you just leave it 20-30 minute in the mixer and it is dissolved again. Then you just add the volume that you need to the media when it is at 50-60 ºC (as you did). You can try in that way...
First you should see your acetosyringone powder that it should not be too old or expired and properly maintain at 4 degree C. If it is good, then you come to preparation. Check whether you are taking absolute DMSO or not. It should be absolute only. Remember it should be prepare freshly all the time
. It should be kept at Room temp once after disolved Iin DMSO, do not freeze it or do not keep on ice once you disolve. Only one ml of DMSO is enough to dissolve 200mg of aceto and you can vortex it for for dissolve. It will never get ppted if you follow the above.
Thank you all for the suggestions. In fact, I placed DMSO-dissolved in -20 degree C after each use. I think that's why it precipitated out from the MS media.
Ms. Patricia, I actually use absolute ethanol to dissolve acetosyringone. You've mentioned about adjusting final volume to 10 ml, did you mean adding dH2O to top up to final volume of 10ml?
AS dissolved in DMSO or ethanol does not precipitate when added to medium at temperature less than 40 degrees Celsius. Maybe your medium was too hot.
We have dissolved Acetosyringone in DMSO & ethanol. It did not precipitate in any of them. But it worked better when dissolved in DMSO! So you may try it out again at RT.
It also depends on your gelling agent. If you are adding it to a tissue culture tested agar it will be fine. If you are using gelrite or other gelling agents, some antibiotics and organics tend to precipitate out. Something to do with the concentration of ion which reacts with the charges on the gelling agent.
Rasika
I believe all the answers indicate to take care of two issues: a. what do you dissolve it in and how much of solvent is used (ethanol vs DMSO), and b. do you freeze stock solution or not? Does freezing the stock solution result in irreversible precipitation? Acetosyringone dissolves fine in absolute ethanol and DMSO both as per the contributors to this question.
I have dissolved Acetosyringone in DMSO . It did not precipitate when used fresh. however problem of precipitation was observed in one month old solution store at 4 degree C
Please read Agrobacterium Protocols by Kan Wang. Acetosyringone will dissolve completely in DMSO only, it is a good cryopreservative.
Thankyou for the contribution and Kudos to ALL.
My question with regards to ACETOSYRINGONE is; how to prepare a 100uM solution using 1M stock ?
Concentration(start) x Volume(start) = Concentration(final) x Volume(final)
i.e) C1V1 = C2V2
For example, if you have 1M stock solution then, take 10 µL (from 1M stock) for 100 mL of media.
Refer the below link for calculation.
https://www.tocris.com/resources/dilution-calculator