5 replies to this topic

[graylox]

Hi all,

with the Pfu in my hands I have the problem that I only get targets amplified with a maximum size of 1kb. Everything above failed. I played around with MgSO4, DMSO, Glycerine, Betaine and BSA, but no luck.
Extension time for a 2kb fragmnet was tested with 1min/kb and 2min/kb.
With Taq I get good results.

Anyone here who has a hint for me what the problem could be?

thanks a lot
Steffen

[vetticus3]

what was the PCR run you used?

[graylox]

The PCR was performed under standard conditions with final Conc. of 0.2 mM each NTP, 250ng each Primer and 5U of Pfu (gives the best results, < 5U decreases yields, > 5U gives no product).

The cycling parameters were:

94°C 2min

30 cycles
94°C 45s
51-57°C 45s (depends on the Fragment used)
72°C 2min

72°C 10min


Once again, works all fine with Taq.

I'm working with Borna Disease Virus which has a highly conserved genome. So you instantly notice every mutation and that’s the reason I don't want to use Taq, 'cause it gives me a lot of mutations.

[Raegan]

i'd say the times you are using aren't long enough.
i use an initial melting time of ~4 min. then at least 1 min for melting, 1-3 min for annealing, and then 2 -3 min for elongation. Pfu is slow. Taq is not.

[Heini]

Hi!
I've used Pfu for 3,9kb fragment. My initial melting time was 3 min in 95C. melting and annealing times were 45 s, elognation time almost 8 minutes (for 4 kb).
I've used only 1,25 units of enzyme.

As Raegan said, maybe you can prolong your times or raise melting temp.

Hope this helps. I've not finished my clonig, but at least the bands from PCR have been right size.

Heini

[badcell]

Hi. I would only increase the extension time. As Raegan and Heini said, Pfu is slower than Taq, it needs around 2 min/kb. I won't change the denaturing or annealing times if they work with Taq.
Also, I normally use 68ºC extension with Pfu, instead of 72ºC. But I'm not sure why, maybe is just some kind of personal voodoo.
Cheers!