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Taq Dilution


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#1 Bassaml7

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Posted 02 September 2009 - 08:05 AM

Hi,

I'd like to ask for instructions about Taq DNA polymerase dilution. To avoid pipetting errors I have to dilute Taq, which is supplied in a concentration of 5 units / microL, to a concentration of 0.5 units / microL . I just wonder if I can use PCR reaction buffer for the dilution process. I know Taq is supplied in a special buffer which contains Glycerol , surfactants , etc . and I would prefer to use a solution of similar composition for dilution but that needs to be purchased and there doesn't seem to be many suppliers for that anyway. So I wonder if I can make a 1:10 dilution using just the PCR buffer and store in aliquotes safely . I am highly concerned about storing Taq in just PCR buffer. I know I could just make a dilution, use the amount I need , then throw away the rest of the solution but I just can't afford that. I need to store the rest for future use.

Thank you in advance.

Edited by Bassaml7, 02 September 2009 - 08:55 AM.


#2 gebirgsziege

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Posted 02 September 2009 - 10:15 PM

Can't you just scale down the vol. used for one PCR reaction (like 0.1 無 instead 1 無)???

Otherwise I think to remember that pequlab supplies their taq with a special dilution buffer.....
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#3 swanny

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Posted 02 September 2009 - 10:22 PM

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
50% Glycerol
0.5% Tween-20
0.5% NP-40
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?
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#4 Bassaml7

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Posted 03 September 2009 - 05:10 AM

Can't you just scale down the vol. used for one PCR reaction (like 0.1 無 instead 1 無)???

Otherwise I think to remember that pequlab supplies their taq with a special dilution buffer.....


Thank you for your reply . I am not sure I understand what you meant but the thing is I don't want to pipette small volumes. The reaction volume for my PCR reaction is 50 無 . If I need 1 unit of Taq then I should add 0.2 無 of the concentrated Taq solution , which is too small volume to deliver precisely .

Edited by Bassaml7, 03 September 2009 - 05:12 AM.


#5 Bassaml7

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Posted 03 September 2009 - 05:49 AM

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
50% Glycerol
0.5% Tween-20
0.5% NP-40
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.


#6 Bassaml7

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Posted 03 September 2009 - 06:23 AM

The delivery of small volumes of Taq is even more and more complicated by the high viscosity of the solution owing to glycerol content.

Edited by Bassaml7, 03 September 2009 - 06:24 AM.


#7 scoob00

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Posted 03 September 2009 - 06:49 AM

Sorry to jump in here, so it's okay to make master mixes with Taq in it? I keep getting told that taq and primers should be the very last things you add, but obviously this is very time consuming. Ready mixes have it all in one tube... Does it depend on what taq you are using?

Cheers.

#8 gebirgsziege

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Posted 03 September 2009 - 07:00 AM

Can't you just scale down the vol. used for one PCR reaction (like 0.1 無 instead 1 無)???

Otherwise I think to remember that pequlab supplies their taq with a special dilution buffer.....


Thank you for your reply . I am not sure I understand what you meant but the thing is I don't want to pipette small volumes. The reaction volume for my PCR reaction is 50 無 . If I need 1 unit of Taq then I should add 0.2 無 of the concentrated Taq solution , which is too small volume to deliver precisely .


depends on your pipet equipment....I am pipetting 0.1無 routinely which works fine. Therefore I would not take the risk of contaminating the Taq with the dilution step.

Do you have a pipet for a vol of 10無, then the 0.2無 should be no problem....

Edited by gebirgsziege, 03 September 2009 - 07:00 AM.

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#9 gebirgsziege

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Posted 03 September 2009 - 07:02 AM

Sorry to jump in here, so it's okay to make master mixes with Taq in it? I keep getting told that taq and primers should be the very last things you add, but obviously this is very time consuming. Ready mixes have it all in one tube... Does it depend on what taq you are using?

Cheers.


I am usually adding the primers but not the taq if preparing lot of MM. Before starting I just add Taq, fill the PCR tubes and then add the DNA. Works good for me.
A man cannot be too careful in the choice of his enemies. (Oscar Wilde)




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