Long Range PCR - (Aug/13/2008 )
I'm doing amplifications of 10-15 Kbp but I have always little efficiency, many problems ... always little products of PCR and the amplifications are not repeated. I am using the kit Long Range from Roche. Someone can give me any suggestions to improve my results?
HI, try finnzymes kits for long PCR, I was on their presentation an it look really good
I never used that kit from Roche. I know Finnzymes kits usually work very well, have a collegue who is crazy about them. I have used Platinum Taq High Fidelity from Invitrogen and Extensor Hi-fidelity PCR enzyme mix from ABgene (now its Thermo), and their both good although personally I always seem to have better results with Extensor. Also, Roche has available application manuals for several techniques, including PCR, which are usually very helpfull.
I used Phusion from NEB for amplification of 11kb from genomic DNA, it is supposed to be highest fidelity on the market. It is incredibly fast too. The problems I had at the beginning were all due to the primers, once that issue was resolved the PCR was very clean and efficient. They say it can do more than 20kb because it has DNA binding domain fused to it, from my results I believe it can do at least 15kb without any problems from genomic DNA.
I love this enzyme for absolutely every challenging PCR!
I used the kit (pfu) from stratagene for my 7 kb plasmid. Every thing is fine.
Thanks! I will try PCRs with these enzymes.
I have a Roche kit also that we've been using for many hundreds of fragments around 8 to 9kb and have never had any problems. It's the Expand Long Range dntPack. Multiple groups here use that successfully. The only problem I've had is trying to do these new pcrs on 16kb fragments...I can't get them to run down the gels!! But I have another topic on that so I won't digress
If you still wanted to get that kit to work, let us know the protocol you're using (reagents, temps, cycles, etc). Are your primers robust?
In our lab we also do long range pcr very routinely- we are using the Expand long range (dNTPack) as well for a long time and we were always happy. In between we tried different other products but the Expand l.r. was always the most robust with best results over a wide range of templates...
Maybe in one assay another enzyme may be little better but altogether the expand is still the best...
but always remember that template dna quality is at least as important as the right enzyme / kit...
hear you soon...
I was busy with other issues and now I return to the long range. I would like to continue doing some more tests with Roche kit (Expand Long PCR). I got amplify, sometimes, a fragment of 13 kb, but PCR is not robust.
I will tell you the conditions under which I am working and I hope you could give me an opinion:
My primers are between 20 and 26 bases and designed in the program Primer3.
The PCR program is:
94ºC – 2 min; 10 cycles: 94ºC – 10s, Ta – 30s, 68ºC – 10-14 min; 25 cycles: 94ºC – 15s, Ta – 30s, 68ºC – 10-14 min; 68ºC – 7 min
The conditions are:
Buffer 1, 2 or 3- 5 ul
dNTPs– 70 umol
Primers– 14 pmol + 14pmol
LongExpandEnzyme- 4,25 U
DNA- 350 ng
Water – until 50 ul