6 replies to this topic

[hannna]

Hi everyone!

I am lately trying to PCR amplify almost my entire plasmid. There is 40 bp distance between my primers. And the whole plasmid is almost 10kb long.
So, I was wondering is this even possible?
I didn't manage to get a product so far. And I'm not sure whether this is just because of the length or because the template itself needs to be longer then the product.
I'm using Phusion High Fidelity MasterMix.

Also, any other tips on long PCR would also be helpful...

Many thanks,

[Papaver]

I've already done this so it should be possible. How long is your elongation time. My experience with Phusion polymerase was that I had to use higher elongation times than for "normal" PCRs, depending on the plasmid. For my latest try to amplify the my plasmid (> 8 kb) I used 6.5 min at 72 °C. Do you use phosphorylated primers? If not, I would try because Phusion prefers them. How many runs do you use?

I'm using Phusion for site directed mutagenesis, amplifying the complete plasmid during 25 runs. I only once checked the product but didn't see any product but I get my clones after transforming into E. coli.
Maybe you could load as much as possible onto the gel just for checking...

[hannna]

Thanks for your reply!

So far I was using 15 s per kb, so 2.5 min.
I'm just now running a pcr with 5 min elongation time, I'll see if it works.
You think I should go even higher?
I have 35 runs.

One of my primers is not phosphorylated and the other one has a digoxigenin label on 5'.
But I used primers like this for shorter fragments. So, they should work with Phusion.

[hannna]

I get a smear when using prolonged extension times.

[Papaver]

PCR sucks...
Unfortunately there are so many komponents you can change; concentrations of dNTPs, primer, template...PCR conditions...
Have you also tried different annealing temperatures? Or look for other polymerases which are disigned for long-fragment PCR.

[Papaver]

Hey,

I was just looking for another Phusion related question and found this in FAQ for "Phusion® High-Fidelity PCR Master Mix with HF Buffer FAQ ":

Q2: I am having trouble amplifying a template that is longer than 5kb. How can I optimize my product yield using Phusion® High-Fidelity PCR Master Mix?

A2: * Use more template. Sample concentration may be too low.
* Template DNA may be damaged. Use carefully purified template.
* Increase number of cycles.
* Lengthen extension time.


Q3: I am having trouble amplifying a template that is longer than 5kb. How can I optimize my product yield using Phusion® High-Fidelity PCR Master Mix?

A3: * Increase template amount. Sample concentration may be too low.
* Template DNA may be damaged. Use carefully purified template.
* Lengthen extension time.
* Increase the number of cycles.
* Lower annealing temperature.
* Titrate DMSO (2-8%) in the reaction.
* Denaturation temperature may be too low. Optimal denaturation temperature for most templates is 98°C or higher.
* Denaturation time may be too long or too short. Optimize the denaturation time.

link: http://www.neb.com/n...roductF-531.asp

Maybe that helps. Good luck!

[tea-test]

is your plasmid GC-rich?
then maybe try the GC-buffer (or try in anyway).
For me that was sometimes a BIG difference. But I only did ~3kb.