SUMMARY:Large PCR products

[Jose_Costoya]

 Hello,

      There is the summary about large PCR products question, I hope this helps
      somebody, thanks very much for all who replied.

                                            Jose

                             Jose-Costoya at seins.usc.es
                             Dpment. of Physiology
                             School of Medicine
                        University of Santiago de Compostela
                                  SPAIN
                             Tel. +34-81-582658
                             Fax  +34-81-574145
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 I've done amplifications of genes of 1700bp using standard protocolls
 (25 cycles, 94deg 40 sec, 50 deg 40 sec, 72 deg 1:30 min)

 (Mix: 2 ul DNA sol., each 2 ul primer (75 uM), 1 ul dNTP mix (10 mM each),
 5 ul 10x polymerase buffer, 38 ul a. dest.; mix, add .5 ul polymerase,
 mix, add 50 ul paraffin oil, spin 5 sec., cycle .....)
 (you can try also adding BSA; thats not required with several polymerases).

 and with the same protocoll fragments of 3250bp; works fine!
 (I KNOW :-) the Dynazyme polymerase from Finnzymes Oy works great!
 but kkep in mind the patent of LaRoche on the PCR process ....)

 Cheers
 SWB

 --

            `oo'
          ( O  O )
             vv \\
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    \\  email:
                      [ Sebastian.Bunka at vu-wien.ac.at ]
                        phone:                   FAX:
                +43-1-71155260          +43-1-7149110
 Location: earth, europe, austria, vienna  Inst. of Bacteriology  Vet.Univ.

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 I've performed PCR the standard way and have obtained around 1.3 kb when
 wanted, no special conditions.

 Try looking into the use of gp90? the use of single stranded DNA binding
 proteins that stabilise it. I read something on this a long time ago. Also
 look into different sources of Taq. I think vent from pharmacia is supposed
 to make up to 12kb (not). Try amersham as well they do a taq also.

 Martin Leach
 --

 .......          Martin Leach                Email:leach at mbcrr.harvard.edu
   _|____      Dept. of Pharmacology       Phone: (617) 638-5323
   / o  /      Boston Univ. School of Med. Fax:   (617) 638-4329
 _/  |-/__==/  80 E. Concord St. (L603)
 (BULLDOZER) \_ Boston MA 02118            "Not the old underpants on your
               USA                           head.....WIBBLE" -BLACKADDER

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 If you have a MacIntosh Computer, I can probably help you.  There are two
 "gopher" programs that can be retrieved from IUBIO.flybase.mac .  They are
 "Amplify and HyperPCR".  With the assistance of these two programs I was
 able to amplify a 4.2 kb fragment from the E. coli genome.  The programs are
 pretty
 much self-explanatory.  Other hints are to reduce the annealing time to 30
 seconds (this cuts down on the smaller nonspecific products) and if at all
 possible use 30 mers for your amplification primers.
                                Best of Luck,
                                Ihor

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 Dear netters,
 
 found the ref to improve yields of long pcr products:


 "Improved yields of long PCR products using gene 32 protein"

 N.A.R.   18:4 1079

 good luck (never tried it)

 Martin Leach

 --

 .......          Martin Leach                Email:leach at mbcrr.harvard.edu
   _|____      Dept. of Pharmacology       Phone: (617) 638-5323
   / o  /      Boston Univ. School of Med. Fax:   (617) 638-4329
 _/  |-/__==/  80 E. Concord St. (L603)
 (BULLDOZER) \_ Boston MA 02118            "Not the old underpants on your
               USA                           head.....WIBBLE" -BLACKADDER

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 From Rasmussen, Utah, U.S.A.:

 I can pass along two references.  The first is "In vitro Amplification of
 DNA fragments > 10 kb" by Kainz et al in Analytical Biochemistry 202,46-49
 (1992).  They recommend Tub polymerase, claiming that it is more processive
 than Taq or Vent.  I have no first hand experience with Tub.

 The second reference is "Effect of Heat Denaturation of Target DNA on the
 PCR amplification" by Gustafson et al in Gene 123, 241-244 (1993).  In this
 paper they use Taq to make DNA in the 2.5 kb range.  They claim that it is
 critical to keep the amount of time spent at the denaturation temperature
 to an absolute minimum.  When they boil their DNA before amplification for
 1 minute they get good yields of large products, when they boil the DNA for
 3 minutes they get almost no product.  Smaller products do not show this
 effect.  They also use an Idaho Technology rapid air thermo-cycler to keep
 the time at denaturation during the amplification at a minimum.

 We use this second protocol and the air-cycler in our lab and we amplify
 1000 to 4000 bp products all the time.

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 I have often used vent polymerase to "primer extend" products in the 4 kb
 range.
 Here at Wash U we have a guy named Wayne Barnes who can extend primers for up
 to 18 kB, using his own enzyme formulation, which he calls KlenTaqLA. I have
 tried this enzyme and found it to work fine on my smaller products. Wayne has
 also found the error rate of his highly processive enzyme to be about that of
 Pfu polymerase, or 12x the fidelity of Taq. Try and reach him for suggestions
 regarding long PCR reactions. his address: barnes at biolgy.wustl.edu
 I have no direct affliation with Wayne or his enzymes. Good luck!
 brett lindenbach
 brett at borcim.wustl.edu

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 Just to add some of my comments to many of your comments regarding
 how to make large PCR products.

 I have had great success in amplifying large fragments.  But
 the caveat is that the template happens to be a plasmid.  When I repeat
 the same procedure using genomic DNA as a template no such luck.
 Of course, in cases where genomic templates seem to be a problem
 a nested set of amplifications have given good results.  All using
 standard (cetus?!) Taq and other condition.

 Is there an opinion concerning amplifying large fragments from
 different templates?  I would love to hear any comments.

 Raj Shankarappa
 bsh at med.pitt.edu

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 I am surprised to see 2.5 kb appear in this discussion. Working from
 human genomic DNA, we regard above 3 kb as starting to get difficult;
 up to 5 kb, you can (almost) always do it with "standard" PCR (ie
 AmpliTaq). Just optimize _everything_ - "touchdown" PCR for maximum
 yield, and titrate Mg and primer concentrations. Try 5 or 10% DMSO -
 if your primers work with it, leave it in. We increase denaturation
 to about 1 min at 920C, and extension to 3-5 min.

 *******************************************************************
 Michael Morris PhD
 Division of Medical Genetics       tel (Switzerland) (22) 702.56.94
 CMU, University of Geneva          fax (Switzerland) (22) 702.57.06
 Geneva, Switzerland                email mike at medsun.unige.ch

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 I amplified a 1.9Kb fragment with standart protocols. You must allow ~1min
 per 1kb for Taq. Amplification was not very efficiant.

        Eitan Rubin.

 Plant Genetics, Weizmann Inst of Science, Rehovot, Israel.
 EMail: bcrubin at dapsas1.weizmann.ac.il
 Tel: (0092)-(8)342421


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