real time PCR machine - (Oct/10/2010 )
so my supervisor is trying to apply for a grant to get a real time PCR machine, and we've been asking for quotes from several different companies. I just started using real time machine pretty much a week ago and really green in knowing of what to get. There are so many of them and each company have different products. I am wondering if you can recommend me of what would be the most robust and easiest machine to get? We need them particularly for gene expression study, so I don't think we need those multiplexing features and all that. I heard that in biorad iQ5 you need to calibrate the machine everytime the volume of reaction changes. Is that true? I am using ABI 7500 now and it doesn't seem that I need to do so, I just need to tell the software the volume of reaction. Nevertheless Biorad iQ5 seems to be pretty popular. Anyhow, if anyone can advice on what features to look for, I'd greatly appreciate it!
no, buy Rotorgene 6000. It is not plate based like ABI, and it doesn't have expensive capillaries like LightCycler from Roche. it is very easy to use. it dominating the market now.
you should consider how many reactions will you likely be performing at one time. I find the ABI machine is great for giving "clean" or "pretty" looking graphs, but the one I have only holds 48 reactions at one time.. which is not that helpful for my purposes. It also takes a longer time to run the qPCR than on some machines that only take an hour.
While Rotorgene 6000 is probably much better in terms of having lower inter-reaction variability, they are much harder for larger sample sizes since conventional things like multichannel pippets don't work so well with them.
7500 is very good. You can do calibration by yourselve (had my time calibrating it to work with EvaGreen).Sometihng to ocnsider is StepOne from ABI - but i dont know the price difference among theose 2..
We have PikoReal in the lab from ThermoFisher. fairly cheap and simple. Plates are cheap as well as they are only 1/4 size and still has 96 reactions. The problem i saw is that the program is slow (u need newer computer for it compared to ABI products) and the white plates are VERY annoying if you use transparent master mix (we used DynamoColor flash, now Thermofisher offers Luminaris blue master mixes - I want to try those).
Rotorgene... well i dont like it personaly - need to cap all the tubes (4 at the time) - its time consuming, and i dont have long pipete tips for them so this machine is kinda used by the people who are worried of NTC going up. Buth the good thing is - the cycles can be VERY short (i got down to 4 seconds) good for some kinetics experiments if you can make them work in qPCR.
I need to do calibration for StepOne of ABI. Can anyone help me to know how to do it without using the company's kit because the kit is so expensive? Thanks.