Do I need Gradient Thermal Cycler? - (Feb/27/2008 )
I am not a molecular biologist, but I am starting a new lab, and foresee the need/desire to do PCR. The application I am interested in is cloning genes expressed in early chick/quail development. What I have learned online and from papers is that we would need to extract/isolate mRNA from the tissues of interest, and then amplify it using PCR. I think we may end up doing this quite often in the future, so am looking into purchasing a thermal cycler. So getting back to the title of the thread - do I need a gradient cycler? In general, what applications would a gradient cycler be used for? To be specific, I am considering the Gradient and Personal versions of the Eppendorf MasterCycler (the latter being about half the cost of the former). Obviously, I would rather save money here, and am leaning toward the non-gradient version.
A gradient option can save a lot of time for optimizing pcr reactions. I have used it for several times already and you just get nicer results because you don't have to trust those dubious primer Tm calculations. also for finding optimal temperatures of PCR-cloning primers it is very useful.
Overall IMHO eppendorf, although the quality appears to be excellent, is maybe not the right company when you are considering saving money
in our institute we have a bio-rad mycycler gradient machine and i can recommend it. it is the easiest machine i have ever worked with, it has a 96 well block for many samples and the gradient option. but I have no idea about the price (list price is 5400.- euros) compared to the eppendorf
If it's an option and you can afford it, get it! I've wasted days optimizing PCR annealing temperatures because we don't have a gradient cycler
h2so4 is correct, if you can afford it, get it. Thankfully, the cost of many cyclers is coming down as the features increase.
If you can't afford one, you can still do many really useful experiments, including touchdown (which takes slightly different Tm's into account) as well as standard PCRs.
I disagree with the above posts. My job is cloning genes (i've literally done hundreds) and i never use the gradient feature on our thermal cycler. I think for double the price it is not worth it. Successful PCR is about so many other things besides optimising primer annealing, if you're relying on a gradient PCR to get your PCR to work then your design is generally poor in the first place. I only ever use the one PCR program - a touchdown from 70 - 60 C with an extension time of 1 min 30 sec and 40 cycles. The main things in PCR (such as yours) are as follows:
- primer design - making sure the Tm is high (i do ~70C) - if the primers fail, optimisation will not dramatically improve your success, design new primers and try different combinations of the old and new primers
- tissue cDNA - important to choose a tissue where your gene is highly expressed (usually the paper characterising the gene has good reliable expression data like northern blots), try a few tissues at once, the expression levels in published data are not always the same as in your samples
- GC% - if your GC% > 55%, do a betaine gradient (0 - 2M final concentration of betaine)
I get about 80% of my genes the first time around using this general philosophy. I usually end up getting my gene pretty quickly if it fails first time around any way - without even thinking about gradient PCR.
Another thing - use biocompare.com to scan what is available to you - excellent website
if you're just starting your lab you might need more equipment besides the thermocycler. i did lots of rt-pcr and never used the gradient, only tried it a couple of times for a multiplex pcr. however these days some lab-mates use the gradient sometimes, if it didn't have that feature i don't think their results would be that much different.
As you are well aware, the gradient feature can be very helpful for optimising PCR conditions. I know it is quite possible to optimise PCR with a normal PCR machine, and I often don't use the gradient, but I'm an old hand at it. I think your purchasing decision should come down to what your priorities are where your expertise lies.
As killerkoz17 already mentioned, someone who is good at PCR can often analyze the sequence of what you're trying to amplify, and design the PCR in such a way that you have a high likelihood of it working without having to resort to using a gradient machine.
That being said, seeing as you are not an expert in cloning, if you can afford a gradient, it might save you a lot of time and effort. PCR takes a long time to master, and the money you invest in the machine might be returned in the long run because of the time you save in not having to repeat PCRs over and over again just to get some sort of amplification product.
Of course, if you can effort, buy. But whether you really need it or not depends if you develop new methods and regularly design new primers, try out new PCR reactions etc., or if you're just doing standard PCRs with everything finished and optimised. I needed it only 2 or 3 times and then I asked in another lab to use their machines. That cost me a question (and an invitation to a coffee one time).
Most PCR machines come with gradient function anyway. And it is not really expensive to get a PCR with gradient function. Gradient PCR is really important. You can have alot of applications with it.
Sorry, but I cannot see why gradient function is so "important". All it does is short-circuit the time to get a product (assuming your primers are good), while giving you a number to use in a report (which isn't actually necessary). A touchdown PCR will produce your product, can be just as easily incorporated into any report, and only uses one reaction tube, rather than the multiple number of tubes required to do a full gradient (12, on my machine).