PCR a fragment of 6kb - (Jun/26/2008 )

I am trying to clone a set of genes and overexpress them in tissue culture

I normally have no problem with cloning genes up to 3kb, but this time for one of the genes, the length is kind of long, it's about 5-6kb

I tried to use invitrogen Platinum Hi-Fi taq, elongation time is 6min, 30 cycles, and got a very weak band, and also the PCR reaction takes forever to finish@@

I wonder how to optimize the condition to get a brighter band such that I would be easier to clone it.

Any suggestions or experiences are welcomed!!

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1. What is the template?
2. you can do several reactions with 30 cycles to get sufficient DNA for cloning. (5 pcr reactions, pool, ppt, clean, clone). No need to get bright band from one.
3. Try other PCR kit.

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1. template is mouse kidney cDNA (TRIZol, then superscript 3)
2. agree, the thing is I am using TA cloning, should be easy, right? But I keep failing to get positive clones when I PCR check clones, so I wonder if imcreasing the concentration of the PCR product (but not the absolute amount of PCR product) will help

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Several points, see if some suit you:

1. Did you check available cDNA clones from AddGene, Open Biosystems etc, to see if it is already available in the form you (a) need it for expt, ( can use for cloning, or © use as a template for PCR.

2. If you know of a tissue expressing higher amount of your gene, try using that as a strarting point. Beware of isofrom differences though if you are ultimately interested in kidney related expts.

3. I am not sure about this one, but generally proof-reading Taqs are not great for TA cloning. You may have to add your own overhangs after PCR if things continue to 'not work'.

4. Change Primers

5. Change PCR Kit.

6. If it is a very specific product, you may do direct PCR clean-up rather than running on gel, that would reduce the agarose and else contamination, while increasing the yield. Cloning would work better that way.

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Now I am thinking about my choice of enzyme

The enzyme I am using is Invitrogen Platinum HiFi Taq, which is not perfect

Any better alternatives?

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No enzyme is perfect. You could try Phusion or PfuUltraII (both fusions of a polymerase with a DNA-binding enzyme, should give higher yields and both are known for high fidelity). However, they do not give you the A-overhang needed for topoTA-cloning, so after purification you should incubate with Taq (in buffer-Mg-dATP) and then use for topo-TA.

But if you get a band, maybe you should lower your annealing temperature after 10 cycles by 2-3 °C. By this time, you should have enough correct template for correct priming, though priming will be more efficient at lower temperature, which should increase the yield.

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for Phusion, the Tm of the primers should be >60°C, the higher the better.
i have good experiences with Phusion. it is also a very fast enzyme, so 3min extension for a 6kb fragment should be enough. i think they recommend 15-30s /kb. then you can also increase the cycle number which is maybe the best and easiest way to get more product.

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wooooow... I agree with Phusion, it's the best choice. But if you cannot clone a 6Kb fragment into a 3.5Kb vector... I assume that you are using invitrogen Topo TA cloning kit or similar... thus the vector is kinda small as compared with your insert hence the absence of positive clones... but I might be wrong... look at this link :

http://www.clontech.com/images/ctq/APR07UP...epPCR_IF_US.pdf

I'd give clonetech InFusion a go...

and since you were talking about being able to clone 3kb frags, I assume you are electroporating as well.

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Phusion is the best polymerase for PCR. Your ligation isn't working well most likely because it is inefficient and you don't have enough insert to get it in there. To get more insert, either perform several reactions as cellcounter suggested or re-amplify your PCR product to get more product. Just keep the 6 minute cycle - it's a long template so it has to be a long extension time - too bad! Reducing the extension time may reduce the amount of product. Also add an additional 2.5 mM MgCl2 to increase primer annealing and perform more cycles to get more product. Given that you are getting a product, albeit a faint one, doing these things will certainly give you more product, which is what you're after.

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Phusion definitely, 3-4 min extension. And as mentioned above, taq incubation to get the overhangs. And, I too would recommend inphusion cloning kit. Always works!

I had no problem getting a 8kb fragment with phusion polymerase.

Cheers

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