Primer Annealing Temperatures - (Aug/21/2012 )
Thanks for all the ideas guys, I ran a gradient and the primer set worked fine at an annealing temp of 60C
Another note: Back in the days when I was still trying to optimise each reaction's Ta, I sometimes run gradient on certain new assays, 55 - 65 Ct, with my hotstart mastermix (HotStarTaq MM from Qiagen), and the result always was that products from all temperatures were equally (very) bright. This happened most of the time I tried the gradient. So I generaly stopped doing that.
Qiagen has a proprietary mix, that often worked for my colleagues when no other polymerase would (not even AmpliTaq Gold), so sometimes just good polymerase can spare you a lot of optimizing time.
Actually in the case of Qiagen is not the polymerase but the simple buffer. Their recipe is the best one. It is a mixture of Tris and MgCl2 at the certain pH/conc that makes that buffer indeed the best Taq buffer on the market (I tested ~10 of them). The funny thing is that the polymerase is the same from all companies and if you change them in the buffer from Qiagen, they will work as well as the one from Qiagen. In my master thesis I purified my own Taq polymerase (in one purification I got 40 mL of what Qiagen sells in 25 uL aliquots) and once we figured out the right buffer recipe, my entire lab used this forever and ever (we did a lot of PCRs; alone I was using 200 uL of Taq polymerase per week).
Depending on the purpose of the PCR, Phusion is far better in amplification than Taq (although more expensive) because of increased processivity due to the extra ssDNA binding domain. On top, Phusion does not make as many errors as Taq if you want to PCR inserts for cloning. And btw for large scale applications is worth cloning Phusion polymerase and purify it (1 L culture leads to 20-30 mL of polymerase after one His-affinity purification step); Phusion is actually Pfu + a ssDNA binding domain from an archea (check patent to see which one)
So, what's the magic Qiagen buffer recipe? Openwetware wants to know.
Unfortunatelly (even though with my entire opensource-science heart I would love to) I cannot give you those recipes because I was working in a company at that moment (confidentiality contract blah-blah) But I can tell you how we identified the candidates and people can also try:
-checked patents (+a couple of Chinese companies who stole recepies from American/European companies) + all known PCR buffer recipies --> extensive google-ing
-did a PCR mastermix/pro buffer (water, dNTPs, buffer, primers, template) with a template of ~1000 bp with normal amount of GC without the polymerase; divided it in 10 tubes (10 uL) each; dilution series of the inhouse purified Taq polymerase 3-5 times diluted in between steps; set up a PCR for 10 steps and ran gels to compare the different buffers (including the commercial Qiagen); this would be a test for processivity since you will get a band only for the first 2-4 dilutions in the series.
-take the best 3 (including Qiagen buffer) from above and do an amplification and cloning and sequence 10 clones each per buffer to get the best fidelity out of the 3 chosen buffers
We did this for both Taq and Phusion and we got the buffers that lead to the same results as the commercial ones.
I know this does not help too much, but maybe there is somebody out here, reading this post and would try the same thing and would be able to post the recipes. I know that I would try the same approach again when I have my own lab (10 years from now ) and share the recipies with the world.
ascacioc on Thu Aug 23 21:51:42 2012 said:
I did a PCR once with primers one at 46 and the other one at 71 (long story, but I couldn't design them better no matter what I did, and I struggled quite a lot). Worked perfectly....but I'm the one with a too complicated PCR program
did you got some amplification ? How did you do that?
see my previous posts, after I said that I did that I also posted a protocol I use for Phusion PCR.
Open source is a nice thing and surely if someones digs out the right composition by trying and googling good for him, on the other hand I have no problem with paying company for a good product they developed
I know this isn't at all timely, but Andreea mentioned SB buffer. The same authors (Brody and Kern) later published on LB buffer (Lithium instead of sodium). Now I run my gels for 10 min at 300V in Owl chambers rated to 150V and everything is great. LB definitely out-performs SB. Also a phusion lover, I'm going to try your touchdown protocol. I've become less-enamored with touchdowns over the last couple of years, but I'll entertain your protocol.