Can I PCR a gene of 2KB? - (Nov/06/2006 )

Hello all, I was wondering if I could PCR a gene of 2Kb, to be more exact it is 1788bps. I know that when your PCR fragment is to big your primers will fall off compromising the fidelity of your PCRed fragment.

Are there any tips in designing good primers, for big fragments? what about the polimerase? is there any good polimerase specifically for long fragments?


I will really apreciate all your help!!!

I've done 5kb before using standard conditions other than lengthening the extension time. I'd try it first with your std conditions, but with perhaps a 2 min extension.

I've done 20kB. 2kb is actually very small. So don't worry.

Polymerase
Use a high fidelity polymerase.

For something small, 2kb to 5kb I use KOD hifi. It is a good polymerase, relatively cheap, with high product yeilds. I would also advice to stay away from "high fidelity Taq". I am biased against any such mutant taq as my labmates have had not but trouble when using such enzymes. Use a proper high fidelity polymerase.

Primers
Make sure the part of your primer that actually binds to the template has identical melting temperatures. I am for a tm of 58 Celcius. I use this temperature because it works nicely with the sequencing protocol that is use in my lab (Extention temperature 60 Celciis).

As a rule of the thumb, the 5'end of your primer should be a little more GC richer then the 3' end of your primer. This bias in GC richness helps as the GC rich 5' end binds holds the primer to the template while the GC poorer 3' end can look around for perfect binding.

I added restriction site to ends of my primers (for ligation purposes), and thus vary the GC content of the primers to a degree by using GC rich guard sequences.

Annealing
Nothing much to say. A standard 4 Celcius lower then the Tm usually works

Extention
On occasion you get better product yeild if you drop the temperature. 70 or 68 celcius rather then the standard 72 Celcius. Increase the extention time to compensate. If you are using KOD hifi, with plasmid DNA 35 seconds is good enough for 2kb. Genomic DNA takes more time about 1 min

Melting
Don't over do it. AT rich DNA doesn't take much to melt. 10 to 15 seconds is good enough for 2kb

Always do a trial run. Always make sure your DNA is of good quality.

If you get no product it is usually the DNA's fault. Dilute the DNA 1:20 to 1:100. Also you can increase the magnesium ion concentration to improve the signal. (However Mg also increases none specific band formation.

best of luck.

Thank you guys for all you're feedback in this matter. I really really appreciate it.