Taq and Pfu polymerase give different amplification - (May/04/2006 )
I try to perform get a 500bp-PCR-Product using Pfu-Polymerase. When I do the PCR with Taq-Polymerase everything works out fine. However when I use Pfu under the same condition, no product shows up. I haven't tried out any other polymerase yet.
1. Has anybody had similar problems? Is there an explanation for this?
2. Can you recommend another polymerase with proofreading-ability?
3. Since the fragment is only 500 bp long I thought about gambling a bit: Just do several batches, use Taq and hope that one resulting fragment is a clean match. What do you think?
4. Why is there only one female in smurf village?
1. I've used severall different polymerases and they all have different optimal conditions dependant on your template and primers. The reason is that they are derived from different bacteria/archaea etc... which have different cytoplasmic concentrations of ions etc, so that's why PCR-buffers for different polymerases as well as optimal cycling conditions will be different.
2. Don't give up yet, you have the Pfu now, try playing around with annealing temp, Mg-concentration etc. Polymerases are too expensive to just throw away after one failure. If it keeps occuring: stratagene has PfuTurbo, PfuUltra and PfuUltraII (optimised versions of Pfu), invitrogen has platinum Pfx, accuprime Pfx and Pfx50, Roche has Pwo and Tgo, Finnzyme has Phusion, NEB has Vent and Deep Vent, there's probably countless others and then you still have all the "high fidelity enzyme mixes" (taq + a proofreading enzyme).
3. That might be a good idea. But, this means you would have to clone them and sequence a lot of colonies which will also be expensie (on the other hand: even if you use a proofreading enzyme: there's no guarantee that no error is incorporated, just the chances of errors are lower, but you would still need to do the cloning and sequencing, but most likely you will get a clean match after a smaller number of attempts). If you would just sequence your PCR product right away: you will very likely see a nice match, because for instance taq incorporates ONE mistake in the first cycle of PCR on just ONE of your template (at a specific position) and you started with for instance 10 template molecules, you will only see this mutation in 10% of your PCR products (assuming that this mistake will not be made a again...), so on the electropherogram you will most likely not notice this one.
4. Smurf's are a bunch of sexists and only need women for procreation? just my 5 cents.
You can use a polymerase cocktail. I have done 10:1 Taq:Pfu. That way, you get the best of both worlds - good amplification from Taq, proof-reading from Pfu.
also, PFU has exonuclease activity. Are you adding the PFU last, on ice and perhaps doing a hostart. This will minimse degradation of your template.
acutally, origninally there were no girls in the smurf village. Gargamel created the smurfette to infiltrate the village, but she felt bad and switched sides to join the smurfs. Does anyone else think that vanity smuf may have been gay?
1. yes. Pfu is pickey. I overcame it by addng more Magnesium, and dmso. Now, with that, i can pfu to amplify things taq won't touch.
2. There are a few out there. The one's i've tried usually flop. Triplemaster seems to work ok.
3. The error rate for Taq is not that high, especially when you consider that you're product isn't that big. You could use Taw, but remember to sequence a number of colonies, just incase.
4. There are 3 female smurfs:
The first was Smurfette, who was created by a magical potion. Her list of ingredients include: "Sugar and spice but nothing nice...A dram of crocodile tears...A peck of bird brain...The tip of an adder's tongue...Half a pack of lies, white, of course...The slyness of a cat...The vanity of a peacock...The chatter of a magpie...The guile of a vixen and the disposition of a shrew...And of course the hardest stone for her heart...."
The second was Sassette. Sassette actually has the same origins as Smurfette (with a minor difference, she wasn't made by Gargamel). The smurflings (Slouchy, Nat, and Snappy) were pitying Smurfette who felt lonely, being the only girl in the village, so they decided to help her. They learned from Papa Smurf that Smurfette was indeed created magically by Gargamel out of clay. They then went to steal Gargamel's formula and magical recipes for creating Smurfette. They successfully created Sassette, who this time didn't need plastic surgery (though her rude behavior and attitude needed a bit of chemical tweaking). As they introduced Sassette to Smurfette, Smurfette was overjoyed at having a female friend, albeit a bit of a tomboy.
The last was Nanny, an old flame of Grandpa Smurf's, Nanny enters the cartoon series after being trapped inside a cursed castle for centuries. Grandpa and the others rescue her, and she makes her home with them in the village.
Wow, you can see where my interests really are.
Ps. vanity gay? never. he was just sensitive, and very handsome.
Check out your extension time and annealing temperature. Pfu polymerase activity is very slow as compared to Taq polymerase and since Pfu prefers sulphate over chloride, you would end up using probably ammonium sulphate in the buffer so you may have to reduce annealing temperature.
That female smurf history is nothing short of amazing...you should publish a review!
I never heard about differences in temperature-specifity but I rechecked annealing-temperature and extension time with a gradient-pcr and it worked.
Thanks for the deep insights into smurf genealogy :-D