Pfu did not work? - (Aug/22/2005 )
I want to PCR a 3kb segment. When I used Tag, it worked fine. Then I changed to Pfu, but got nothing, no band at all. I also divided 100ul Pfu PCR reactin into 10 pcr tubes and used gradient Tm, still got nothing. What happens? Any suggestions?
Thanks a lot.
Pfu needs about twice the time that taq requires. Change the extension times to suit your needs, it's about 2 min per kb.
If it is standard pfu, it indeed needs about twice the time taq needs. Apart from that, did you check Mg-concentration, quality of your DNA (run a PCR with taq as a positive control?).
Thanks a lot.
You can also "dope" your reaction with regular Taq to get more efficient extensions. The Pfu is used for its proofreading ability, but sometimes it works too well! It may never get to the end of the 3kB product during a given cycle. If you add some Taq to the mix it will increase the processivity of the enzyme, but still give you the proofreading you want. The exact ratios of Taq/Pfu are a best guess anyway when you buy the enzyme premixed. Good luck!
I used PfuUltra and the extension time is 4 min. Pfu reaction buffer offers a final Mg+ concentration of 2mM. I may try to increase the extension time and increase Mg+ concentration to 3mM.
Is there any other PCR you can do to check your pfuUltra?
Is there any way to check the quality of your DNA? (redo reaction with taq?)
I'm working with PfuUltra myself every now and then and for this one there is no need to increase the extension time (Stratagene's protocol says 1 kb/min and I use that time which works for me). If you're doing PCR @ 68°C for extension you might benefit from a longer extension time, in the protocol for Accuscript-RT-PCR kit, it says that @ 68°C you need 3 min/kb. (although I recently did a site directed mutagenesis with extenstion temp of 68°C and used 1 min/kb).
PfuUltra for me is the best proofreading enzyme I have worked with, concerning yield and sensitivity.
Why is pfu so 'sluggish' is it the temperature or ?
I was using normal Pfu for my PCR but wasn't getting decent results so I switched to PfuTurbo, which has Taq mixed in it. I still wasn't getting decent results until one of our post-docs told me that Cl will severly diminish Pfu's activity, so I switched to MgSO4 for adding Mg to the reaction. Things work pretty well now for me.
Also, I've been running at ~2min/kb @ 68C with a final 10min extension phase to help clean up the reaction.