It's a Micro World after all is a blog dedicated to discussing pretty much whatever I feel like. When I delve into scientific matters it will primarily be discussing microbiology (agricultural, bioenergy, and environmental focus). Otherwise, I'll probably ramble on about sports and life.
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I imagine that most scientists are creatures of habit, and some superstition as well. Speaking from experience, I know I have protocols which I've worked with for a decade or more and I'm loathe to change or tweak them. They work, why reinvent the wheel? This same thought process extends to reagents that are typically used in the lab. There were certain manufacturers that I "grew up" using, and remained loyal to that particular brand throughout my graduate and postdoc work. When putting my own lab together, while I was willing to negotiate some* on equipment**, by and large I was more than happy to stick with the "tried and true" microbiological and molecular reagents that I had used for years. For instance, I would never consider buying my restriction enzymes from anywhere but NEB. They work, why switch?
Problem is, projects change, which means conditions change. This point was eloquently brought home to me this past week. We had received some free Taq. We were told that in our system, this Taq would most likely perform better than any other Taq we could find on the market. For general amplifications we use what I would consider a "middle of the line" (in terms of cost) Taq which has worked pretty well in our hands. After all, Taq is Taq, no? Now there were instances where we wouldn't get amplification like we expected, and those were frustrating. To avoid this, we adjusted our extraction protocols to get cleaner DNA. We were told that this Taq would allow us to skip that extra extraction. I didn't believe it but told my support scientist to go ahead, skip the second extraction, and try our standard Taq and this new one side by side. Here are the results ...
After looking at this comparison, I doubt I'll ever say "Taq is Taq" again. The first set of reactions is the test Taq we received. The second set of reactions is our routine Taq. While our Taq worked in 7 of the 8 reactions, they were faint. The new Taq reactions worked like gangbusters! Additional tests reconfirmed these findings. Needless to say, we're switching.
So, I get not wanting to switch things up, especially when things are rocking and rolling. However, if you have the time to do a comparison here and there, especially in areas where things are working but might just be scraping by, it might benefit you in the long run.
* I do not believe all thermal cyclers are manufactured equal, but I could care less if I have an Ohaus, Sartorius, or Mettler Toledo balance. Likewise I don't give a rats patooie who manufactures my vortex or microcentrifuge.
**Unless it involved BioRad, which I refuse to purchase anything from.
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What Taq is that?! We've been having amplification issues in my lab for a while and have been experimenting with several different enzymes, but with random results (i.e., sometimes the test out performs our standard, but then the next sample it doesn't). I definitely want to give your stuff a try.
And yes, I have been known to cling to a particular brand or manufacturer b/c 'if it ain't broke, don't fix it.'
There are certain reagents that I will use over and over again because I have validated them myself or seen someone's else's validation. But for the most part, I could give shit where I get my DNaseI from, its going to work. And I agree with you on the thermocyclers. The best one I ever used was a decrepit and aging Perkin Elmer, I almost wanted to steal it and take it with me when I left the lab. I and many others are also quite partial to their pipetors.
Come on, TJ, you can't make a post like this without outing the two Taq's! We use the Finnzyme Phusion here in lab, mainly because that's what our Illumina sequencer uses for generating clusters. I don't know if it's as efficient as other Taq's, but it has the highest fidelity.
I didn't out them because I imagine that different reagents will work in different systems, differently.
For example, I'm not going to out the Taq which had a bit of a dismal performance under the conditions we placed it under for our test. We purposely skipped a second extraction, which when performed, allows this Taq to work admirably. Plus, it's served me well for a long time, but the systems I study are MUCH different now than when I started using it. Since they're a major biomedical reagent maker, I doubt they had environmental molecular biologists in mind.
As for the new Taq, it's the KAPA2G FAST HotStart Taq. We loves it and it works well in our current system. If we have to make radical changes to our protocols (for whatever unforeseen reason) it may wind up being a less viable solution (costs outweigh the return). As it is now, cutting out that second kit to use our current Taq decreases our costs considerably which will more than offset any increase we see in buying this Taq (I haven't even compared the prices yet to be honest ... I know it's going to cut down on man hours which is savings enough at the moment).
The worst is when you go to a new lab, and their default is different than what you are used to.
Part of the problem with testing is if you are using precious samples. You may not be getting the best yield, but you can get predictable yeilds, which, is sometimes what you need.
Geeka, very true on the "precious samples" comment. More often than not, I am fortunate enough that I always collect more than is necessary. Since some of my work is temporal, I can never go back and capture that sample again (e.g., there will only ever be one Jan 27th, 2011).We'd been running with the predictable yields line of though for the above work for awhile.
If it comes down to low yields, I have been known to tell the rep that very thing. Hey, testing out your enzymes is going to cost me time and money in other reagents. Usually they have a sample prep kit which they'll throw in (to get me to use their enzymes). I'm then more than happy to show them side by side comparisons, report to them yields, etc on the kits/enzymes we've put through the paces. I also let them know the characteristics of our samples because it may help them tailor what they recommend to me in the future as well.
Well, Taq is often Taq plus a proofreading polymerase or an antibody to make it hot start. So pure Taq is probably pure Taq, but it's hard to find these days.
For whatever reason, when I work with plasmodium DNA (it's 80% AT rich; weird stuff, really) it HATES Pfu and most gold shiny polymerases. The promega GoTaq seems to work pretty well for it though. I had come out of a cancer lab that was obsessed with SNP genotyping, so the idea of using a non proofreading polymerase had horrified me. It wasn't that I was married to a partiuclar brand, but the notion that regular Taq was much better? It just didn't feel right.
My lab is very aprticular about the reagents we use. There is a central services unit in my building that makes up common reagents but over time we have stopped buying things from them so that we can use OUR SDS or OUR HEPES etc. No idea if it has had any real effect but I seem to get better results than aome of my other fellow PhD students, but I do have to make up all my own reagents whereas they don't have to :(
James, I remember having to make up a reagents, some of which had over seventy compounds in them. Not only that but they needed to be made as sub-reagents. Some were only soluble in EtOH, some needed to be heated whereas others couldn't be heated. It was a total pain in the rear. Usually took you an entire day to get the entire thing made up. With luck, you could get a few experiments out of a particular batch. What matters the most is your results. I always believed that if you made up your own reagents and you got great results, it was worth it. So while your fellow PhD students bought their base reagents, but got lousy results (forcing them to redo experiment after experiment), you probably saved time and money in the long term by doing it all on your own (and correctly) the first time.
Typically the only stock solutions I allow my lab to buy are RNA reagents. It's much easier to purchase them than to DEPC-treat solutions (given that I have a tiny autoclave too), and in this case I ONLY use Ambion.
Best of luck in your research!