8 replies to this topic

[guido53]

Hi all, I've to buy a new taq high fidelity to amplify up to 8kb in human genomic dna. which one do you recommend me? I found so many enzymes and all are so "perfect"(obviously), I hope you could help me.

[pDNA]

i like a lot Finnzymes Phusion Polymerase ...but as always its more or less a matter of taste

Regards,
p

guido53, on 18 May 2011 - 09:00 AM, said:

Hi all, I've to buy a new taq high fidelity to amplify up to 8kb in human genomic dna. which one do you recommend me? I found so many enzymes and all are so "perfect"(obviously), I hope you could help me.

[bob1]

guido53, on 18 May 2011 - 09:00 AM, said:

Hi all, I've to buy a new taq high fidelity to amplify up to 8kb in human genomic dna. which one do you recommend me? I found so many enzymes and all are so "perfect"(obviously), I hope you could help me.

I think you mean a new polymerase (Taq is one type of polymerase, isolated from Thermus aquaticus), Taq itself is never high fidelity but it does come in some mixes with other proof reading enzymes which enable high fidelity in the reaction.  

Anyway, enough of my ranting... I recommend KOD polymerase from Takara, I have found phusion to give relatively weak bands.

[guido53]

yes sorry, i mean a new polymerase, you are right....In the takara site i found TaKaRa LA Taq™ Hot-Start Version, is it the KOD polymerase?
I found also the phusion, accuprime pfx by invitrogen,long amp by neb, Long PCR —Advantage® Genomic LA by clonetech and AccuTaq™ LA DNA Polymerase by sigma.....but the takara enzyme seems to be better....which is the size of the longest amplicon you obtained with the kod?have you used it for a sequencing?
Thank you very much

[phage434]

It was a five years ago, but at the time i had very good luck with the Roche Expand enzyme for doing long PCR amplifications.  I would probably try Phusion first at this point.

[bob1]

guido53, on 18 May 2011 - 11:43 PM, said:

yes sorry, i mean a new polymerase, you are right....In the takara site i found TaKaRa LA Taq™ Hot-Start Version, is it the KOD polymerase?
I found also the phusion, accuprime pfx by invitrogen,long amp by neb, Long PCR —Advantage® Genomic LA by clonetech and AccuTaq™ LA DNA Polymerase by sigma.....but the takara enzyme seems to be better....which is the size of the longest amplicon you obtained with the kod?have you used it for a sequencing?
Thank you very much

Sorry, I meant Novagen, but had just had someone asking me about takara stuff.  KOD is called KOD polymerase, but there are other forms such as KOD XL.  It is also available from EMD/Merck.  I have used it for not particularly long products or about 1500 bp, but it should work up to 6 kbp.  It is very fast - 15 s/kbp c.f. Taq which is 1 min/kbp.  I have sequenced the products and found them to be fine.

[guido53]

my target is 6-7 kb long, until now i used platinum pfx by invitrogen but unsuccessfully...now i'll study better the phusion, the kod and the roche expand enzyme...@ bob1 sorry but I don't understand what you said about the takara enzyme i found.....
thank you very muche for the help

[Adrian K]

bob1, on 19 May 2011 - 05:24 PM, said:

Sorry, I meant Novagen, but had just had someone asking me about takara stuff.  KOD is called KOD polymerase, but there are other forms such as KOD XL.  It is also available from EMD/Merck.  I have used it for not particularly long products or about 1500 bp, but it should work up to 6 kbp.  It is very fast - 15 s/kbp c.f. Taq which is 1 min/kbp.  I have sequenced the products and found them to be fine.

I do agree with Bob1, I use KOD hotstart and KOD xtreme hotstart in my work, up to 5kb. It works, although I did experienced a few substitution error.

[epimaster]

Hi everybody, If anybody knows how I can make long PCR product from long cDNA that I have? I'm looking for a taq polymerase that can polymerase Long product-around 4Kb-from long  cDNA,if any helpful idea?  

Thanks alot.