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Lowered extension temps with Phusion


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#1 Warren

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Posted 19 September 2009 - 06:26 AM

Has anyone tried using lowered extension temps with Phusion polymerase, and if so, what did you use for a new estimate of the time needed for extension? For example, Finnzymes recommends 15-30s per kb under normal conditions (72 degrees) -- did you allow more extension time, and if so how much with lowered extension temps? Thanks! Warren..

#2 fishdoc

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Posted 19 September 2009 - 06:47 AM

Has anyone tried using lowered extension temps with Phusion polymerase, and if so, what did you use for a new estimate of the time needed for extension? For example, Finnzymes recommends 15-30s per kb under normal conditions (72 degrees) -- did you allow more extension time, and if so how much with lowered extension temps? Thanks! Warren..



I haven't tried it, but I think someone in our lab has. I happened to see one of her programs the other day, and I think it had an extension time of 65 C. If I remember, I'll ask if she does anything differently.

#3 phage434

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Posted 19 September 2009 - 11:53 AM

You would expect the reaction rate to be twice as slow when the reaction temperature is reduced by 10C, but it probably is not that bad. There is little downside, except for waiting, to making the extension time longer. I'd use 1 min/Kb.

#4 Warren

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Posted 20 September 2009 - 08:32 AM

You would expect the reaction rate to be twice as slow when the reaction temperature is reduced by 10C, but it probably is not that bad. There is little downside, except for waiting, to making the extension time longer. I'd use 1 min/Kb.


Thanks! I FINALLY got the sequence to amplify with Phusion hot-start. These are yeast mitochondrial (non-coding) sequences that are very A-T rich. I could get amplification with iTaq, although I had to tweak the annealing temps, but nothing with Phusion. With just the lowered extension temps I got a smaller band (at least it was something) then by adding some DMSO as well, I am at least able to get nice amplification of the correct sequence, although at this point I still have some smearing and non-specific bands. I basically pick GC-rich areas to make my primers, but the problem is alot of the sequences look similar in different places in the mtDNA....so my high Tm primers (designed for Phusion hot-start) I think were getting some non-specific binding, hence the DMSO is probably helping by reducing the non-specific primer binding at the lower temps. I guess I find it strange that iTaq could polymerize through AT regions at 72 and Phusion seemingly can't.

Originally I thought that some of the smaller bands I get are non-specific binding, but now I wonder if its the polymerase "jumping" across AT rich sequences. What do y'all think? Warren..

#5 phage434

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Posted 20 September 2009 - 12:01 PM

Normal extension temperatures do not work for very high AT regions. I discovered this the hard way, but then discovered that this was a known effect. See PMID 8628694,
Su96, Reduced extension temperatures required for PCR amplification of extremely A+T-rich DNA. I would try Betaine instead of DMSO.

#6 Warren

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Posted 21 September 2009 - 06:08 AM

Normal extension temperatures do not work for very high AT regions. I discovered this the hard way, but then discovered that this was a known effect. See PMID 8628694,
Su96, Reduced extension temperatures required for PCR amplification of extremely A+T-rich DNA. I would try Betaine instead of DMSO.


Thanks, I had actually gotten the idea from a few of your old posts, and had subsequently read that paper....I am surprised that this isn't included in literature for polymerases! I am curious though, why would you try betaine rather than DMSO? I'll have to order some betaine, the DMSO I already had. BTW, for whatever reason, it seems like I could get iTaq to polymerize through this AT rich region but not Phusion, which is also weird. Warren..

#7 Warren

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Posted 12 October 2009 - 07:48 PM

Normal extension temperatures do not work for very high AT regions. I discovered this the hard way, but then discovered that this was a known effect. See PMID 8628694,
Su96, Reduced extension temperatures required for PCR amplification of extremely A+T-rich DNA. I would try Betaine instead of DMSO.


Thanks, I had actually gotten the idea from a few of your old posts, and had subsequently read that paper....I am surprised that this isn't included in literature for polymerases! I am curious though, why would you try betaine rather than DMSO? I'll have to order some betaine, the DMSO I already had. BTW, for whatever reason, it seems like I could get iTaq to polymerize through this AT rich region but not Phusion, which is also weird. Warren..


Just as a follow-up, in case anyone reads this thread for advice in the future: the temperature I ended up using with the Phusion Hot-Start is 64.5 (in this case I am just doing two cycle amplification, 98 and 64.5). It seems as though 1 min per kb in this case is not quite enough, for a 1.5 kb fragment 2 min worked well. Of course there is no separate annealing step so the 2 min includes annealing as well. Anyway, Phusion will actually amplify these AT rich regions extremely well at 64.5, just gotta give it enough time to do the extension. Warren..

#8 alland

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Posted 20 March 2010 - 11:35 AM

Warren's suggested cycling parameters worked for me using Phusion.

98oC 60s
98oC 20s
64.5oC 11 min
repeat step 2 and 3 for 24 more cycles
64.5oC 11 min
Hold

Primers were 44 nt and 78oC Tm (as determined by the finnzymes tm calculator)

I was able to amplify an Arabidopsis 8.1 Kb promoter with 70% AT. My reaction conditions were identical to the 50uL reaction specified in the product insert except I used 2U or 1uL of phusion (instead of the suggested 1 U or 0.5 uL).




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