Amplify 4.5 kb pcr product without using special polymerase - (Nov/25/2005 )
Hi everybody,
Could you answer to my question: if it is possible to amplify 4,5 kb DNA fragment without special commercial kit for long pcr product?
I used Pfu polymerase and elongation time from 4 to 8 minutes, recommendation is 2 min for each 1 kb, but without product.
I tried to change annealing temperature from 40 to 60 °C, but without any positive (also any unspecific) results. My primers are 32 and 31 nt long with Tm 59,9 and 60,1°C.
I lost 3 months with optimalization of pcr and I see that without kit it is too difficult to get positive results, but I want to verify it before ordering the kit.
Sasen
hi
sometimes you can amplify 4.5kb with one enzyme and not with one other. It's not always a long PCR extension kit needed...
I worked with Pfu and it didn't gave me results for one exp. Phusion did the job.
Surprisingly, for an other similar size amplification, it was the contrary...
In other exp none gave me results good. Eppendorf triple master did the job very well. (actually, succesfully amplifie 7kb with it too)...
Hi Fred33,
Thank you for your answer.
Sasen 
hi,
i've having similar problems.
check this papers
Barnes, W.M. 1994. PCR amplification of up to 35 kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc. Natl. Acad. Sci. USA 91: 2216-2220.
3. Cheng, S., Fockler, C., Barnes, W.M. and Higuchi, R. 1994. Effective amplification of long targets from cloned inserts and human genomic DNA. Proc. Natl. Acad. Sci. USA 91: 5695-5699.
wish me luck. i'm going to try this tomorrow.
best,
carla
Unless your time is worthless just buy a Long PCR kit - three months wasted on a PCR is more than long enough!
Could you answer to my question: if it is possible to amplify 4,5 kb DNA fragment without special commercial kit for long pcr product?
I used Pfu polymerase and elongation time from 4 to 8 minutes, recommendation is 2 min for each 1 kb, but without product.
I tried to change annealing temperature from 40 to 60 °C, but without any positive (also any unspecific) results. My primers are 32 and 31 nt long with Tm 59,9 and 60,1°C.
I lost 3 months with optimalization of pcr and I see that without kit it is too difficult to get positive results, but I want to verify it before ordering the kit.
Sasen
Hi Sasen:
Why don't you try other kits or DNA polymerase? Try the LA from Takara or DyNAzyme EXT DNA Polymerase from NEB? and in addition, I think may be you should better to check the GC content of your fragment of interest? May be some additive such as DMSO, Betaine shuold be added if the GC content is rather high!
best regards, Good luck, may God bless you!
Pfu polymerase can require very different conditions in my experience. If you have trouble amplifying with it, try a mixture of regular taq with the pfu. To answer your question in the topic, yes it is very possible to get 4.5 kb on sequence using a taq polymerase. It can vary from template to template however.
i think it is a difficult thing to amplify 4.5kb fragment by Pfu.if you don't require high fidelity of PCR production,you can try mixture of Taq and Pfu or exTaq from NEB.it works well in my lab!
If you really need the high fidelity of Pfu, go for PfuTurbo or PfuUltra from stratagene, have used ultra for SDM up to 9 kb, worked fine.
Hi,
Definitely possible. I used a standard proofreader Accuprime and got a 6Kb product just today. Just tried it thinking that it wouldn't work, but got a nice product. I guess it is the way things go in the lab when you think things should definetly work they never do and vice versa.
Scott