how to amplify a long gene? - (Mar/16/2007 )

hi everyone
recently i want to amplify a 8.6kb gene from the total gemonic DNA. throught twice PCR, i just can get a dim line under the UVP.
Could you tell me some improvement or some details which i must pay attention to?

Well I guess you could attack this problem from several different angles

The first would be the polymerase.
So first off what high fidelity polymerase are you using?
Phusion? KOD long template etc... Not all proof reading polymerase mixes are equal, there are formulations for long PCR product. With the exception of phusion, the magic ingredient to most long template PCR polymerase mixes is the addition of thermostable uracil DNA glycosylase, whcih you can add yourself.

One of the causes of failure for long range PCR is the denova production of dUTP (from themal deamination of dCTP). When dUTP is incoperated into the growing DNA strand, it causes the polymerase to pause and repair the remove the bad base. However during the pause period there is a probility of the polymerase falling off, and thus terminating the extention. And as you know, purely on kinetics, PCR favour the shorter product, which soon become very numerous.

Now aside for polymerase, you should make sure your DNA is as clean as you can make it.

The next thing you could do is to play about with the PCR cycling conditions. I will need to know your primer sequence to make any kind of recommendation. However, a trick you could do is to decrease your extention temperature from 72 Celsius to 68 Celsius, increasing extention time to compensate. The lower temperature decreases the probability of the polymerase falling off.
Also don't over do with the melting time, too much and you will start degrading your polymerase.

You could also add additives, like Mg ions, K+ ion, ammonium ions, to improve activity of the polymerase (the down side is you get more side none specific bands coming up)

You could also add glycerol (1% - 5%) to improve stability of the polymerase (thus activity). Betaine (0.1M - 2M) and DMSO to reduce annealing temperature.

Let see, and if all else fails go for bulk. If you are currently running one vial of 50ul... try running 8 vials of 50ul. So you get more product.

As you are seeing a band, you are already having a succesful PCR, so be glad as that isn't easy for longer templates.

The tricks and considerations made by Perneseblue are what I would focus on also. Another trick is to decrease your annealing temperature after 10 cyles in your second PCR, during the first you have specific priming and later on by decreasing your temp (by 2 degress or so) you get more product without losing specificity as you already have "relatively much" correct primer binding sites.

thanks very much!
i will think more about the angles.