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DNA Polymerase for PCR (pfx-invitrogen)

DNA Polymerase for PCR (pfx-invitrogen) - PCR - Polymerase Chain Reaction Forum

DNA Polymerase for PCR (pfx-invitrogen) - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #1  
Old 03-04-2009, 06:27 PM
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Default DNA Polymerase for PCR (pfx-invitrogen)



Hi, I wanted to know if anyone has used less than the recommended amount that invitrogen suggests for their PFX Tag DNA polymerase. The product insert for a 50ul PCR reaction states to use about 1 unit or approximately 0.4ul of pfx? We currently use this amount for our long PCR and our DNA targets are GC rich on the order of about 1500 to 5000 basepairs.

Has anyone used less than the recommended volume in their master mix for a 50ul reaction to amplify the above-mentioned product lenghts and still achieve the right size yield without observing secondary bands? This would be helpful to know. Thanks
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  #2  
Old 03-13-2009, 05:53 PM
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Default Re: DNA Polymerase for PCR (pfx-invitrogen)

I haven't used Pfx Invitrogen, but I've used Pfu polymerase from other brands. My general opinion is that for Pfu, it's much trickier than regular Taq. The brands I've used suggest varying amounts, and I find that it's generally between 1.25 - 2.5 U (depending on the enzyme concentration).

I'm not entirely sure bout the 1 unit recommended for Pfx. That is actually lower than the amount recommended for Pfu by other brands. For GeneRacer (Invitrogen), the recommended amount of Pfx is 1.25 U (0.5 ul in 50 ul reaction), whereby it is for a type of difficult PCR.

You could try using 1 U, but I would suggest increasing it slightly to 1.25 U if you're not getting any reactions. Plus if you're adding any additives (eg. DMSO, betaine, etc), you definitely need a higher amount as these additives partially inhibit Pfu activity.

In general, for myself I use 1.25 U of Pfu for a no-frills PCR, and 2.5 U of Pfu when I'm adding in additives or I believe it's a difficult template.

p/s: I hope you're not doing 50ul PCR reactions. Pfu isn't cheap, and I usually run 15 ul reactions if it's a new PCR or I'm not certain of the results.
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Old 06-16-2009, 08:33 PM
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Default Re: DNA Polymerase for PCR (pfx-invitrogen)

Pfx has a very active proof reading activity. I've used much less (about half of recommended) and seen improvements of amplification. Also reducing extension time, while keeping recommended enzyme units, has helped me. I've noticed that if I have lower template concentration, this condition gives the Pfx the chance to degrade the primers. So you can either play around with the concentration of the enzyme, the template or the primers, or the extension time.

Also, when amplifying from a low concentration template, a gene on a plasmid for example, adding filler DNA (an empty vector, which you know does not contain sequences that might be amplified by the primers) helps boost the amount of PCR product. This suggests to me that the extra DNA help to sequester the exo activity.
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Old 06-16-2009, 08:43 PM
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Default Re: DNA Polymerase for PCR (pfx-invitrogen)

Pfx has a very active proof reading activity. I've used much less (about half of recommended) and seen improvements of amplification. Also reducing extension time, while keeping recommended enzyme units, has helped me. I've noticed that if I have lower template concentration, this condition gives the Pfx the chance to degrade the primers. So you can either play around with the concentration of the enzyme, the template or the primers, or the extension time.

Also, when amplifying from a low concentration template, a gene on a plasmid for example, adding filler DNA (an empty vector, which you know does not contain sequences that might be amplified by the primers) helps boost the amount of PCR product. This suggests to me that the extra DNA help to sequester the exo activity.
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Old 01-13-2012, 08:44 AM
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Default Re: DNA Polymerase for PCR (pfx-invitrogen)

i would appreciate to have your comment:
i prepared the mastermix using following componenets: 10X pfx amplification buffer 5 ul, 10 mMdNTP mixture 0.75 ul, 50mM MGSO4 0.5ul, primers 10pmol each, DNA template 1.5 ul,platinum pfx DNA polymerase 0.2 ul, water 15.05 ul and end volume is 25 ul. thermocycler program is 94 C 3 min, 94 30 s, 54 30 s, 72 C 30 s, 72 C 7 min, 8 C forever, total number of cycle 35.
But I did not get any amplification. Do you find any faults over thsi protocol? or do you have any suggestion how I should try next time?I would be thankful for your reply.
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Old 01-14-2012, 02:32 PM
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Default Re: DNA Polymerase for PCR (pfx-invitrogen)

Quote:
Originally Posted by vetsurya View Post
i would appreciate to have your comment:
i prepared the mastermix using following componenets: 10X pfx amplification buffer 5 ul, 10 mMdNTP mixture 0.75 ul, 50mM MGSO4 0.5ul, primers 10pmol each, DNA template 1.5 ul,platinum pfx DNA polymerase 0.2 ul, water 15.05 ul and end volume is 25 ul. thermocycler program is 94 C 3 min, 94 30 s, 54 30 s, 72 C 30 s, 72 C 7 min, 8 C forever, total number of cycle 35.
But I did not get any amplification. Do you find any faults over thsi protocol? or do you have any suggestion how I should try next time?I would be thankful for your reply.
The protocol seems fine, but it's hard to say without proper troubleshooting and optmization.

My suggestion is to run using a regular Taq. If you can get the band with regular Taq, then it means your primers etc are working fine. Then you can move on to trying with Pfx.
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Old 01-19-2012, 08:31 AM
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Default Re: DNA Polymerase for PCR (pfx-invitrogen)

does pfx polemerase work well in extention temperature 72 C or should i strictly lower to 68 C as recommended by the manufacturer.
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  #8  
Old 01-20-2012, 01:29 PM
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Default Re: DNA Polymerase for PCR (pfx-invitrogen)

Quote:
Originally Posted by vetsurya View Post
does pfx polemerase work well in extention temperature 72 C or should i strictly lower to 68 C as recommended by the manufacturer.
I can't say with certainty, but based on my experiences with the annealing temperature with Pfu and Taq, e.g. for the same sequence using the same primers;
Taq: anneals at 60,
Pfu: anneals at 56

I'd suggest following the manufacturer's instructions on Pfx. Typically I set the extension at 70 C for Pfu compared to 72 C for Taq.
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Old 08-20-2012, 10:38 AM
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Old 11-26-2012, 07:15 PM
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Default Re: DNA Polymerase for PCR (pfx-invitrogen)

Hi everybody
I have the same problem with pfx,at first i wanted to amplify 5 kb fragment from mouse genomic DNA,but i didn't get any band.i divided it to 2400 and 2600 bp and agian no band was seen.I could amplify them bay Taq but ai sould amolify them by a proofreading enzyme like pfx.
Would you please help me?
Thanks a lot.
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