second-strand buffer - Will someone tell me if these calculations are correct? (Aug/21/2008 )
I've been thoroughly spoilt by readymade reagents in my former lab and would appreciate any help as I go about the exciting process of making my own for my new project.
This is what I need to make:
100 ml of 10X second-strand cDNA synthesis buffer (500mM Tris-HCl pH7.8, 50mM MgCl2, 10mM DTT)
This what I have:
1M Tris-HCl, pH8.0
DTT crystals (MW 154.3)
MgCl2 hexahydrate crystals (MW 203.31)
This is what I propose to do:
Dilute 50ml of Tris-HCl with 40 ml of DEPC-treated water, adjust pH to 7.8 with conc. HCl, make up volume to 100 ml, dissolve 0.1543 gm of DTT and 1.0166 gm of MgCl2, 6H20 and autoclave.
Are my calculations correct? Any advice would be greatly appreciated.
This is what I need to make:
100 ml of 10X second-strand cDNA synthesis buffer (500mM Tris-HCl pH7.8, 50mM MgCl2, 10mM DTT)
This what I have:
1M Tris-HCl, pH8.0
DTT crystals (MW 154.3)
MgCl2 hexahydrate crystals (MW 203.31)
This is what I propose to do:
Dilute 50ml of Tris-HCl with 40 ml of DEPC-treated water, adjust pH to 7.8 with conc. HCl, make up volume to 100 ml, dissolve 0.1543 gm of DTT and 1.0166 gm of MgCl2, 6H20 and autoclave.
Are my calculations correct? Any advice would be greatly appreciated.
You could do it that way and your calculations are correct. Assuming here that you don't need that much buffer to make cDNAs, I would instead probably make up stock solutions of each ingredient and then make a small (1 ml) tube when you need it. Hence, I would take some of the 1M tris and pH it down to 7.8, make up 500 mM MgCl2 (10.16g/100ml) and 1MDTT (1.5g/10 ml). Then make up your solution: 500 ul tris, 100 ul Mgcl2, 10 ul DTT, 390 ul H2O. This may seem like more work initially, but all of these solutions are very common and would probably be used for something else at some point. Hence, you would actually be saving yourself time in the long run.
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You could do it that way and your calculations are correct. Assuming here that you don't need that much buffer to make cDNAs, I would instead probably make up stock solutions of each ingredient and then make a small (1 ml) tube when you need it. Hence, I would take some of the 1M tris and pH it down to 7.8, make up 500 mM MgCl2 (10.16g/100ml) and 1MDTT (1.5g/10 ml). Then make up your solution: 500 ul tris, 100 ul Mgcl2, 10 ul DTT, 390 ul H2O. This may seem like more work initially, but all of these solutions are very common and would probably be used for something else at some point. Hence, you would actually be saving yourself time in the long run.
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Thanks, smu2, your advice is greatly appreciated. Looks like you've been doing this for a while....