long PCR Thermocycler

[Dr. Duncan Clark]

In article <5balcl$rbq at rzsun02.rrz.uni-hamburg.de>, soenke behrends
<behrends at plexus.uke.uni-hamburg.de> writes
>
>Hello methods-reagents people,
>I have a question for "long PCR specialists":
>Can you recommend any special Thermocycler for long PCR?
>Is in your opinion the choice of the Thermocycler critical?
>I want to set up long PCR for the first time. 
>So I would also be happy about hints which enzyme (or kit) 
>to choose, whether thin tubes are vital and whether the choice
>and quality of primers is any different than in regular
>PCR. Has anyone tried the capillary air cycler for long PCR?
>

We are developing a long PCR enzyme mix and have done a fair amount of
playing with those parameters. Basically thin walled tubes are a must;
hot start helps or alternatively keep template and primers away from the
enzyme and mix the reagents just prior to starting (keep on ice until
then); each make of machine differs and it is likely PCR conditions will
need to be played with; Magnesium optimum is quite critical so be
prepared to adjust; design your primers such that they have very similar
tm and make it close to 68C then you can just denature and
extend/anneal; use a machine that allows an autoextend ie after say ten
cycles add an extra 20 secs on the extension time on cycle 11 another 20
secs on 12 etc.; 10-15kb is not too difficult, beyond that a lot of
playing can be necessary. We are using PE machines because they
consistently give results. 

In a nut shell long PCR is not yet robust. We have tried umpteen very
different Thermus polymerases mixed with various proof-reading enzymes
at various ratios and they all behave differently. Most will go up to
15kb but beyond that only the odd mix works. The choice of reaction
buffer (not Mg but actual reagents ie Ammonium sulphate or KCl or K
acetate, etc) is critical as to whether a mix works or not. It's a real
pain!! If you would like to beta test a mix email me.

Good luck 

Duncan    

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The problem with being on the cutting edge is that you occasionally get 
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Dr. Duncan Clark
DNAmp Ltd.
TEl/FAX 01252376288
http://www.dnamp.com
http://www.genesys.demon.co.uk