Posted 25 July 2013 - 04:06 PM
Posted 28 July 2013 - 09:00 AM
Posted 01 August 2013 - 03:03 AM
Great question. Had the same discussion with my PI here.
In my old lab we used a mini-mix, now I make them as individual reactions and add the 2.5ul of cDNA to the samples. I actually have to say that with the later method I get way less variation and it takes half the time to do.
Would be interesting to hear what people say about this.
In our genomics centre they tell you to make mini-mixes and also their robot for the 384 pipettes triplicates out of a 96 well plate.
Posted 01 August 2013 - 07:18 AM
Well I'm not a real-time expert but in my view a "mini-mix" is not a replicate for the template amount and master mix variability and any possible errors/variability occurring here, as it's all the same but divided by three. Only the effects that may come from other sources of variability such as well position or pcr machine variation and all "possible issues during amplification" you can control with this.
Anyway if the latter points are major sources of variability, then I'd say it's useful to do these mini-mixes, if not then I'd perhaps prefer "real" replicates with each template added singly (and if template amount differences are a major source of variability that needs to be controlled).
One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.
Posted 01 August 2013 - 10:48 AM
Okay, thanks! This was pretty much in line with what I was thinking. I've been setting up individual reactions and will continue to do so.