Taq Dilution

[Bassaml7]

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.

[jiajia1987]

Hmm...I have yet to dilute my Taq DNA polymerase at all, but I don't see why you can't just dilute it in molecular grade water.

Let's see what the rest says.

[jonoave]

I'm extremely curious to know what kind of PCR reaction you are doing that requires you to dilute your Taq. Taqs are generally supplied in 5U/ul, and you generally use less than 0.05 unit in a PCR reaction.

Eg: In a 50 ul reaction mix, add in 0.5 ul of Taq(5U/ul).
Whereby 5U x 0.5 ul / 50 ul = 0.05 units.

I usually use less than that, around 0.037 units with perfectly good results. However, if you do insist on diluting it, I would think it's best you ask the manufacturer/supplier if they have dilution buffers (I know one company which provides dilution buffers for their Pfu). If not, I think that it's better if you use the 10x buffer to dilute your Taq. Do it in small amounts in many aliquots to prevent using the same tube of diluted Taq repeatedly and undergoing thawing/freezing.

[Bassaml7]

Originally Posted by jonoave

I'm extremely curious to know what kind of PCR reaction you are doing that requires you to dilute your Taq. Taqs are generally supplied in 5U/ul, and you generally use less than 0.05 unit in a PCR reaction.

Eg: In a 50 ul reaction mix, add in 0.5 ul of Taq(5U/ul).
Whereby 5U x 0.5 ul / 50 ul = 0.05 units.

I usually use less than that, around 0.037 units with perfectly good results. However, if you do insist on diluting it, I would think it's best you ask the manufacturer/supplier if they have dilution buffers (I know one company which provides dilution buffers for their Pfu). If not, I think that it's better if you use the 10x buffer to dilute your Taq. Do it in small amounts in many aliquots to prevent using the same tube of diluted Taq repeatedly and undergoing thawing/freezing.

Nothing special at all, just a typical PCR.
If I add 0.5 ul to 50ul reaction volume then that means I am delivering 2.5U to the reaction volume . It's known that the optimal amount ( not concentration) ranges from 0.2 to 2 units . The number you calculated , which is 0.05 , is a concentration not an amount . And it's a rather high concentration for Taq . Too much Taq may result into smear in Gel electrophoresis due to amplification of non-specific products . 2.5 units / 50 ul may be acceptable , but , still , 0.5 ul is a small volume to deliver , especially that Taq is supplied in a solution containing 50% glycerol which means it has high viscosity which further complicates pipetting small volumes .

[jiajia1987]

Hmm true, that is basically what I do too. I just add 0.5uL to get 0.05units if I need that amount. Have never done any dilutions for my Taq. You can take jonoave's suggestion of asking the supplier/manufacturer.

[jonoave]

Originally Posted by Bassaml7

Nothing special at all, just a typical PCR.
If I add 0.5 ul to 50ul reaction volume then that means I am delivering 2.5U to the reaction volume . It's known that the optimal amount ( not concentration) ranges from 0.2 to 2 units . The number you calculated , which is 0.05 , is a concentration not an amount . And it's a rather high concentration for Taq . Too much Taq may result into smear in Gel electrophoresis due to amplification of non-specific products . 2.5 units / 50 ul may be acceptable , but , still , 0.5 ul is a small volume to deliver , especially that Taq is supplied in a solution containing 50% glycerol which means it has high viscosity which further complicates pipetting small volumes .

Thanks for this! I knew there was something I was missing when typing my response, but I couldn't remember what it was. Yeah, Taq is usually quoted in units and not concentration. My bad...

Taq is highly sensitive; the one I'm using was prescribed as 1-2.5 units. I tend to use less the lower range, which is around 1.5 units. For visualisation, I use 0.1 ul of Taq for a 15 ul reaction and have consistent results.

As for pipetting Taq, I generally put it in the Mastermix, whereby I'll pippette a larger volume. I usually leave out the dNTPs or template out of the Mastermix, which is generally in a higher amount than Taq and is easier to pipette individually. I've never had any problems with this, and I find that Taq is hardly a factor in any failed PCRs.

Erm, so basically I just think that diluting Taq is not a good idea... :P

[Bassaml7]

Originally Posted by jonoave

Thanks for this! I knew there was something I was missing when typing my response, but I couldn't remember what it was. Yeah, Taq is usually quoted in units and not concentration. My bad...

Taq is highly sensitive; the one I'm using was prescribed as 1-2.5 units. I tend to use less the lower range, which is around 1.5 units. For visualisation, I use 0.1 ul of Taq for a 15 ul reaction and have consistent results.

As for pipetting Taq, I generally put it in the Mastermix, whereby I'll pippette a larger volume. I usually leave out the dNTPs or template out of the Mastermix, which is generally in a higher amount than Taq and is easier to pipette individually. I've never had any problems with this, and I find that Taq is hardly a factor in any failed PCRs.

Erm, so basically I just think that diluting Taq is not a good idea... :P

I see, so I think preparing Mastermixes is the best practise . Thank you for your response .

[jyaron]

Mastermixes is generally what my lab does.