need suggestions about using Pfu Turbo - (Aug/08/2005 )
Hello Guys,
I'm doing PCR using Pfu Turbo. My template is in pcDNA3.1, about 2.7 kb, and not GC rich.
My PCR reaction is set as: 94C 2min, 94C 30sec, 52C 30sec, 72C 3min ( or 6min, 35 cycles), 72C 7min, 4C
Unfortunately, Pfu never works out for me, while normal Taq gives me a low yield under the same condition.
Anyone can give me a suggestion to make Pfu working?? I really need the high fidelity for cloning. Thanks in advance
Hi,
do you use the appropriate buffer? I use Pfu Turbo too and it works though the vial seems to be quite old. Do you use Taq- buffer for your Pfu?
If so it might be necessary to increase Mg-concentration up to 3 mM, though your PCR might become more unspecific. As your template is a plasmid this shouldn´t be a problem I think.
My suggestion would be to get some µl of the 10 x cloned Pfu buffer.
Cheers,
good luck
Hi Bomber, Thanks for your advice.
Of course I'm using the Pfu Buffer. And I've tried to enhance my Mg concentration up to 6mM, but it didn't work.
do you use the appropriate buffer? I use Pfu Turbo too and it works though the vial seems to be quite old. Do you use Taq- buffer for your Pfu?
If so it might be necessary to increase Mg-concentration up to 3 mM, though your PCR might become more unspecific. As your template is a plasmid this shouldn´t be a problem I think.
My suggestion would be to get some µl of the 10 x cloned Pfu buffer.
Cheers,
good luck
Hi bigeye,
If you've already optimised for Mg and annealing temperature then I suggest checking your dNTPs. Use a fresh batch if possible.
You could also try using a Pfu-Taq cocktail (50:50 mixture) to see if you can get any amplification, though if you're not getting a strong band with Taq, it may be a moot point. I'd suggest optimising for a strong band with Taq before moving onto Pfu (ie, fresh reagents/primers, change annealing temp, Mg conc., etc), since Pfu is a tad trickier to get going. Also make sure that you're not using too much DNA in your reaction - do you have any other primers in your plasmid that you can check the reaction with (generic primers, like T7/SP6 - I'm not familiar with your plasmid)?
It sounds to me like the reaction isn't optimised, or else you'd have a strong band with Taq. Hopefully it's just one of your reagents, and you can chuck it out. If not, then it may be your primers - a re-design may be necessary!
Good luck,
Nicole 
If you've already optimised for Mg and annealing temperature then I suggest checking your dNTPs. Use a fresh batch if possible.
You could also try using a Pfu-Taq cocktail (50:50 mixture) to see if you can get any amplification, though if you're not getting a strong band with Taq, it may be a moot point. I'd suggest optimising for a strong band with Taq before moving onto Pfu (ie, fresh reagents/primers, change annealing temp, Mg conc., etc), since Pfu is a tad trickier to get going. Also make sure that you're not using too much DNA in your reaction - do you have any other primers in your plasmid that you can check the reaction with (generic primers, like T7/SP6 - I'm not familiar with your plasmid)?
It sounds to me like the reaction isn't optimised, or else you'd have a strong band with Taq. Hopefully it's just one of your reagents, and you can chuck it out. If not, then it may be your primers - a re-design may be necessary!
Good luck,
Nicole
Thank you Nic_T, what I am using is the Pfu Turbo mastermix. And to rule out the possibility the mastermix is not good, I used another batch. Neither of them worked. And about the template inhibition, it should not be possible, because I lowered the amount of plasmid down about 10, 100, 1000 fold, still nothing. But I'll try to optimize the condition for Taq and then move on to Pfu, as you suggested.
Hi,
Pfu turbo can be picky sometimes. I made the same experience. However, I recently tested TAKARA ExTaq polymerase and was pretty impressed by the results. You can get a free sample if you go to the TAKARA webpage. The enzyme is in between Taq and Pfu in terms of the error rate and you can clone your PCR directly into a T/A vector.
Good luck.
Freiberger
If you've already optimised for Mg and annealing temperature then I suggest checking your dNTPs. Use a fresh batch if possible.
You could also try using a Pfu-Taq cocktail (50:50 mixture) to see if you can get any amplification, though if you're not getting a strong band with Taq, it may be a moot point. I'd suggest optimising for a strong band with Taq before moving onto Pfu (ie, fresh reagents/primers, change annealing temp, Mg conc., etc), since Pfu is a tad trickier to get going. Also make sure that you're not using too much DNA in your reaction - do you have any other primers in your plasmid that you can check the reaction with (generic primers, like T7/SP6 - I'm not familiar with your plasmid)?
It sounds to me like the reaction isn't optimised, or else you'd have a strong band with Taq. Hopefully it's just one of your reagents, and you can chuck it out. If not, then it may be your primers - a re-design may be necessary!
Good luck,
Nicole
Thank you Nic_T, what I am using is the Pfu Turbo mastermix. And to rule out the possibility the mastermix is not good, I used another batch. Neither of them worked. And about the template inhibition, it should not be possible, because I lowered the amount of plasmid down about 10, 100, 1000 fold, still nothing. But I'll try to optimize the condition for Taq and then move on to Pfu, as you suggested.
Thanks. That's a good news to me, but I can't find the link for free samples!!
Pfu turbo can be picky sometimes. I made the same experience. However, I recently tested TAKARA ExTaq polymerase and was pretty impressed by the results. You can get a free sample if you go to the TAKARA webpage. The enzyme is in between Taq and Pfu in terms of the error rate and you can clone your PCR directly into a T/A vector.
Good luck.
Freiberger
If you've already optimised for Mg and annealing temperature then I suggest checking your dNTPs. Use a fresh batch if possible.
You could also try using a Pfu-Taq cocktail (50:50 mixture) to see if you can get any amplification, though if you're not getting a strong band with Taq, it may be a moot point. I'd suggest optimising for a strong band with Taq before moving onto Pfu (ie, fresh reagents/primers, change annealing temp, Mg conc., etc), since Pfu is a tad trickier to get going. Also make sure that you're not using too much DNA in your reaction - do you have any other primers in your plasmid that you can check the reaction with (generic primers, like T7/SP6 - I'm not familiar with your plasmid)?
It sounds to me like the reaction isn't optimised, or else you'd have a strong band with Taq. Hopefully it's just one of your reagents, and you can chuck it out. If not, then it may be your primers - a re-design may be necessary!
Good luck,
Nicole
Thank you Nic_T, what I am using is the Pfu Turbo mastermix. And to rule out the possibility the mastermix is not good, I used another batch. Neither of them worked. And about the template inhibition, it should not be possible, because I lowered the amount of plasmid down about 10, 100, 1000 fold, still nothing. But I'll try to optimize the condition for Taq and then move on to Pfu, as you suggested.
94C 2min, 94C 30sec, 52C 30sec, 72C 3min ( or 6min, 35 cycles), 72C 7min, 4C
change your reaction time to:
95C 30sec 95C 30sec 55C 1min 68C 8min(35 cycles) 68C 20min 4C -
this works for me everytime. when I design, I try to make the TM close to 55C of both primer, 54C or 56C is fine. the homolog part at least 15bp.
Yizhou He
I really like using a mix of Pfu and ExTaq.
The proofreading activity of the Pfu will correct any mistakes made by the ExTaq.
I usually set up three rxns. 25%ExTaq/75% Pfu; 50%/50%; and 75%/25%.