qPCR with different genes - (May/02/2015 )
Hello,
I am planning to set up my qPCR plate but i have 24 different genes so i have 24 different primers which i want to run in triplicates along with 2 housekeeping genes. I want to know how can i set up my qPCR plate without a lot of pipetting as i think i have to make different master mix for each gene and then pipette them individually.
How many samples? With 26 genes you won't fit all of the the genes on one plate even if you had two samples only.
(for relative quantification I suppose) You need to have all samples within a gene on the same plate. You first need to calculate that and the plate design.
And of course you need different mastermix for each gene. But there is no option that would make such big experiment "without a lot of pipetting", unless you have a multichanel pipette.
Trof on Sun May 3 21:01:23 2015 said:
How many samples? With 26 genes you won't fit all of the the genes on one plate even if you had two samples only.
(for relative quantification I suppose) You need to have all samples within a gene on the same plate. You first need to calculate that and the plate design.
And of course you need different mastermix for each gene. But there is no option that would make such big experiment "without a lot of pipetting", unless you have a multichanel pipette.
Yes, i too think the same.
So, lets say i take 2 genes and have 24 samples for each gene then i can make it in triplicates. Yes, i have a mulit-channel pipette so i will make use of it. Any other suggestions to minimize errors?
Mad researcher on Sun May 3 21:14:14 2015 said:
Trof on Sun May 3 21:01:23 2015 said:
Yes, i have a mulit-channel pipette so i will make use of it. Any other suggestions to minimize errors?
And of course you need different mastermix for each gene. But there is no option that would make such big experiment "without a lot of pipetting", unless you have a multichanel pipette.
To minimize error - don't use the multichannel, these are notoriously variable across the tips, with (usually) the outside tips picking up less than those closer to the centre.
Good multichannels don't have that problem. But it is even more essential with multichannels to look at the pipet tip volume for each channel, to verify that things are working. This is a habit that you should of course also use when working with single channel pipets.
@Mad researcher
are you really planning to pipet something or are you designing some weird RPG? In that case, use a good pipetting robot, well serviced. That is the most convenient and error-free way how to pipet real-time plates. Done. Game over.
In a thought experiment it is difficult to to weight different things that may affect the precision and comfortability in reality, because in reality you are often limited by what you do and don't have and by what you do and don't need.