Protocol Online logo
Top : New Forum Archives (2009-): : DNA Methylation and Epigenetics

5' aza-cytidine - stock solution - (Feb/01/2010 )

Hi all,

I'm gonna start some dnmt inhibitor experiments. Can someone tell me how to make a stock solution ? how stable it is ?? i have 10 mg solid 5 aza2dc from sigma chemicals. thanks in advance.

-RKA-

RKA on Feb 1 2010, 07:07 AM said:

Can someone tell me how to make a stock solution ? how stable it is ?? i have 10 mg solid 5 aza2dc from sigma chemicals.


Hello!

For our 5Aza, we use the following as a stock:

100,000X Stock
-Dissolve 1 mg of 5-Aza-2dc in 4.380 mL of STO* media (w/o FBS)
-Aliquot in sterile microfuge tubes, 50Ál per tube
-Store in -80
no idea how stable, but at -80 I'd say at least a year

Then for the working solution we use:

Working Solution (1X=0.01ÁM)

-Make a 1:10 dilution (10,000 X) by taking 10Ál of 100,000X stock and adding 90Ál of STO media
-Make another 1:10 dilution (1,000X) by taking 10Ál of 10,000x stock and adding 90 Ál of STO media
-Make another 1:10 dilution (100 X) by taking 10Ál of 1,000x stock and adding 90 Ál of STO media
-Finally, make another 1:100 dilution by taking 10Ál of 100x stock and adding 990 Ál of STO media

*STO is what we use as a cell culture media.
-100mL Glasgows MEM
-1.2mL of 200mM L-Glutamine
-1.2mL of 100mM Sodium Pyruvate
-1.1mL DMEM

-k_undertoe-

thanks for the answer, everyone using different solvent to dissolve 5AZA, like ethanol, dmso etc...doo u think that doesent matter ??



k_undertoe on Feb 1 2010, 07:07 PM said:

RKA on Feb 1 2010, 07:07 AM said:

Can someone tell me how to make a stock solution ? how stable it is ?? i have 10 mg solid 5 aza2dc from sigma chemicals.


Hello!

For our 5Aza, we use the following as a stock:

100,000X Stock
-Dissolve 1 mg of 5-Aza-2dc in 4.380 mL of STO* media (w/o FBS)
-Aliquot in sterile microfuge tubes, 50Ál per tube
-Store in -80
no idea how stable, but at -80 I'd say at least a year

Then for the working solution we use:

Working Solution (1X=0.01ÁM)

-Make a 1:10 dilution (10,000 X) by taking 10Ál of 100,000X stock and adding 90Ál of STO media
-Make another 1:10 dilution (1,000X) by taking 10Ál of 10,000x stock and adding 90 Ál of STO media
-Make another 1:10 dilution (100 X) by taking 10Ál of 1,000x stock and adding 90 Ál of STO media
-Finally, make another 1:100 dilution by taking 10Ál of 100x stock and adding 990 Ál of STO media

*STO is what we use as a cell culture media.
-100mL Glasgows MEM
-1.2mL of 200mM L-Glutamine
-1.2mL of 100mM Sodium Pyruvate
-1.1mL DMEM

-RKA-

i don't think it matters. i dilute it in DMSO because my vehicle contains DMSO as well. also, i thought i saw on the sigma site that it's only stable for a month at -80.

RKA on Feb 2 2010, 02:43 AM said:

thanks for the answer, everyone using different solvent to dissolve 5AZA, like ethanol, dmso etc...doo u think that doesent matter ??



k_undertoe on Feb 1 2010, 07:07 PM said:

RKA on Feb 1 2010, 07:07 AM said:

Can someone tell me how to make a stock solution ? how stable it is ?? i have 10 mg solid 5 aza2dc from sigma chemicals.


Hello!

For our 5Aza, we use the following as a stock:

100,000X Stock
-Dissolve 1 mg of 5-Aza-2dc in 4.380 mL of STO* media (w/o FBS)
-Aliquot in sterile microfuge tubes, 50Ál per tube
-Store in -80
no idea how stable, but at -80 I'd say at least a year

Then for the working solution we use:

Working Solution (1X=0.01ÁM)

-Make a 1:10 dilution (10,000 X) by taking 10Ál of 100,000X stock and adding 90Ál of STO media
-Make another 1:10 dilution (1,000X) by taking 10Ál of 10,000x stock and adding 90 Ál of STO media
-Make another 1:10 dilution (100 X) by taking 10Ál of 1,000x stock and adding 90 Ál of STO media
-Finally, make another 1:100 dilution by taking 10Ál of 100x stock and adding 990 Ál of STO media

*STO is what we use as a cell culture media.
-100mL Glasgows MEM
-1.2mL of 200mM L-Glutamine
-1.2mL of 100mM Sodium Pyruvate
-1.1mL DMEM

-assilem-

Hi,

I am currently using Sigma 5-AZA that was diluted as described in the post above in PBS in april 2009. It still works fine...

Tobi

-tobikenobi-