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Making a stock solution for my drug using DMSO

cAMP cGMP DMSO Cholesterol Efflux Macrophage

Best Answer Missle, 15 October 2013 - 05:31 AM

Yes, 10mg in 1ml would be ~20mM and 5ul into 1ml (a 200x dilution) would give a conc. of 0.1mM.

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#1 Frazermb

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Posted 15 October 2013 - 03:38 AM

I am about to start work on a project.

 

I will be using cAMP and gAMP analogues to stimulate cholesterol efflux in macrophages.

I have to prepare my drugs to a concentration of 0.1mM in DMSO.

 

The stock of each drug can have no more than 5ul per 1000ul of DMSO.

 

The MWt of the cAMP is 430.09 and the cGMP is 500.14.

 

The testing will use 20ul of stock drug into 1ml of media containing macrophages.

 

The cGMP I has come on a bottle in powder form (10mg), looking at it I will probably get about 2mg out to use.

 

So if I wanted to make a stock solution to say 1mM I would weight out 500.14mg and add it to 1ml of DMSO?

 

So when using my stock at 0.1mM I would make a 1:10 dilution from 1mM stock?

 

Sorry if this is basic I’m just getting confused and probably over complicating things.

 

Thanks

Frazer



#2 mdfenko

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Posted 15 October 2013 - 04:02 AM

So if I wanted to make a stock solution to say 1mM I would weight out 500.14mg and add it to 1ml of DMSO?

 

So when using my stock at 0.1mM I would make a 1:10 dilution from 1mM stock?

500.14mg/ml=1M. 500.14mg/L=1mM=500.14ug/ml

 

you are correct that you dilute 1mM 10x (1ml to 10ml) to make 0.1mM.


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#3 phage434

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Posted 15 October 2013 - 04:17 AM

If you have only 10 mg in the bottle, then I would strongly suggest that you not attempt to weigh small amounts. Far better is to add liquid to the bottle and dissolve the entire amount, in the original bottle, then move that solution (now at a known concentration) o a container of your choosing.



#4 Frazermb

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Posted 15 October 2013 - 04:25 AM

 

So if I wanted to make a stock solution to say 1mM I would weight out 500.14mg and add it to 1ml of DMSO?

 

So when using my stock at 0.1mM I would make a 1:10 dilution from 1mM stock?

500.14mg/ml=1M. 500.14mg/L=1mM=500.14ug/ml

 

you are correct that you dilute 1mM 10x (1ml to 10ml) to make 0.1mM.

 

Thanks for the speedy response.

 

The drug I got is in powder and I will probably use about 2mg of this.

 

If I weight out 2mg and add it to 1ml DMSO that will give me a final concentration of 4mM?

 

Then if I wanted to use 20ul of 0.1mM for my experiment I would dilute my stock x40 (1ml in 40ml ) to make 0.1mM?

 

What's got me confused also if that we have been told that we cannot use any more than 5ul of DMSO per 1ml of media and in our well for the experiments it will have 1ml of media containing macrophages.



#5 Frazermb

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Posted 15 October 2013 - 04:26 AM

If you have only 10 mg in the bottle, then I would strongly suggest that you not attempt to weigh small amounts. Far better is to add liquid to the bottle and dissolve the entire amount, in the original bottle, then move that solution (now at a known concentration) o a container of your choosing.

thanks for responding, I to was thinking that but when speaking to one the the lab techs they said I would be lucky to get 2mg out of the bottle.

 

I too was thinking its best to add solution to the bottle and use all 10mg and have a known concentration stock. I'm not sure why they are thinking of weighing out 2mg. 



#6 Missle

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Posted 15 October 2013 - 04:35 AM

I'm confused around the restriction you mentioned of "The stock of each drug can have no more than 5ul per 1000ul of DMSO".  Does this mean your stock solution can have no more than 0.5% DMSO?  Or can your working solution (once you've added your drug to your cells) not contain more than 0.5% DMSO.  If either of those are accurate, I don't think your plan falls within those restrictions.....



#7 Missle

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Posted 15 October 2013 - 04:38 AM

Sorry, I didn't see some of the new replies before I responded.  Depending on the solubility of your drug, either make a more concentrated stock solution to get around the 5ul DMSO per 1ml media or initially make a concentrated stock with DMSO and then dilute it to your desired stock concentration with media or buffer.  DMSO can cause major artifactual results sometimes, which is probably the reason for the restriction.



#8 mdfenko

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Posted 15 October 2013 - 04:47 AM

If I weight out 2mg and add it to 1ml DMSO that will give me a final concentration of 4mM?

 

Then if I wanted to use 20ul of 0.1mM for my experiment I would dilute my stock x40 (1ml in 40ml ) to make 0.1mM?

yes, 2mg in 1ml will make ~4mM, but phage434 is correct, when you have so little it is best to just add solvent to the bottle and use the whole thing (if the bottle has not previously been opened).

 

if you have a 4mM stock then 1ml made to 40ml will be 0.1mM.


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#9 Frazermb

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Posted 15 October 2013 - 04:53 AM

Sorry, I didn't see some of the new replies before I responded.  Depending on the solubility of your drug, either make a more concentrated stock solution to get around the 5ul DMSO per 1ml media or initially make a concentrated stock with DMSO and then dilute it to your desired stock concentration with media or buffer.  DMSO can cause major artifactual results sometimes, which is probably the reason for the restriction.

Hi again thanks for responding.

 

Yes I too was wondering about this as if the initial stock is to be made up using DMSO. Dilution of the stock with media or a buffer will probably be needed. Yes that is the reason for the restriction. I will be doing a viability assay (MTT) using my drug solution prior to any testing. 



#10 Missle

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Posted 15 October 2013 - 04:58 AM

That makes sense.  Then your stock solution (in DMSO) has to be at least 200x concentrated >20mM.



#11 Frazermb

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Posted 15 October 2013 - 05:00 AM

 

If I weight out 2mg and add it to 1ml DMSO that will give me a final concentration of 4mM?

 

Then if I wanted to use 20ul of 0.1mM for my experiment I would dilute my stock x40 (1ml in 40ml ) to make 0.1mM?

yes, 2mg in 1ml will make ~4mM, but phage434 is correct, when you have so little it is best to just add solvent to the bottle and use the whole thing (if the bottle has not previously been opened).

 

if you have a 4mM stock then 1ml made to 40ml will be 0.1mM.

 

Yes sorry 1ml made up to 40ml.

 

I was thinking that if the drug is 10mg in bottle I could add 20ml solution (possibly DMSO) to make a 1mM stock?

 

Then if I wanted a 0.1mM solution for my experiments I would take 1ml and make it up to 10ml ? 



#12 Missle

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Posted 15 October 2013 - 05:09 AM

Is your 10mg bottle large enough to hold 20ml?  Why not make a more concentrated solution say a 10mM or 20mM stock and then take 100ul or 50ul to make your 10ml solution.  It will minimize the effects of your solvent....



#13 Frazermb

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Posted 15 October 2013 - 05:09 AM

That makes sense.  Then your stock solution (in DMSO) has to be at least 200x concentrated >20mM.

So to make a 20mM stock I would need to add just 1ml DSMO to 10mg of drug?



#14 Frazermb

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Posted 15 October 2013 - 05:21 AM

Is your 10mg bottle large enough to hold 20ml?  Why not make a more concentrated solution say a 10mM or 20mM stock and then take 100ul or 50ul to make your 10ml solution.  It will minimize the effects of your solvent....

Probably not now that I think about it. I think it may well be a approx 5ml, maybe. 

 

So if I make a 20mM stock I could use 1ml DMSO. I could then take 5ul and make it up to 1ml (buffer or media) to have a concentration of 0.1mM which I could then use 20ul for my experiment?



#15 Missle

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Posted 15 October 2013 - 05:31 AM   Best Answer

Yes, 10mg in 1ml would be ~20mM and 5ul into 1ml (a 200x dilution) would give a conc. of 0.1mM.







Also tagged with one or more of these keywords: cAMP, cGMP, DMSO, Cholesterol Efflux, Macrophage

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