I would like to suggest you try making master-mixes of reagents rather than dilute the Taq down to 0.5 U/ul. This spares the small pipetting volumes (and associated errors you mention). If you really want to dilute the Taq, the data sheets that come with it should give the recipe for the storage buffer. This is the NEB Taq storage recipe:
10 mM Tris-HCl
100 mM KCl
1 mM Dithiothreitol
0.1 mM EDTA
pH 7.4 @ 25蚓
Personally, I'd think it was more trouble than it's worth, compared with making a mastermix, unless you just do 1 or 2 reactions at a time. You must be doing small reaction volumes, if you only need 0.5 U/ul, yes?
Thank you for your reply . I already thought about the Mastermix idea , but I am just doing 1 or 2 PCR reactions daily. Plus I am still doing the optimization step so it's early for preparing mastermixes because If I prepare them now then I'll probably use only one or two portions of each mastermix and throw away the rest of it .
No, the reaction volume I am doing is the typical volume , 50 無 . And to add 1 unit to each reaction volume I need to add 2 無 of a 1 : 10 diluted solution of Taq which is far better than adding 0.2 無 of the concentrated solution . I just don't know why companies supply Taq in such a high concentration . And yet most of them don't supply a dilution buffer with the enzyme , or don't even bother making one . We all know that we usually need just 0.2-2 units of Taq for a standard 50 無 reaction volume . So what's the point in making such a concentrated stock solution of Taq ? I mean 0.5-1 units / 無 seems ideal. Why 5 units / 無 ???.....perhaps the reason is the need to dilute those ingredients in the recipe you mentioned to the maximum extent to spare their effects on the PCR reaction .
I am already aware of the recipe you provided . Glycerol is usually added as an antifreeze because Taq should not be allowed to freeze during storage . The remaining surfactants are stabilizers as far as I know. I guess I can't just store a diluted portion of Taq in PCR buffer . There are sure reasons why this recipe contains all these ingredients . I guess I have no choice but to look for a dilution buffer supplier .
Even If I prepare mastermixes , I should use all of them . Because these don't contain glycerol or the other stabilizing surfactants ( or rather they contain a diluted amounts of the components in the recipe ) so Taq may freeze in the mastermix . I am not sure if the diluted amounts of the ingredients coming from the stock Taq solution are enough to protect Taq . It's really puzzling . I wonder what do you guys do ? so you just prepare a mastermix and use it up ?
Edited by Bassaml7, 03 September 2009 - 06:06 AM.