I have never done a qPCR experiment before so I need help with designing one.
The attached is the layout of a 48-well plate that I have designed. I am measuring mRNA expression during a 48-hour period using 'No treatment' as a calibrator. The method I am using is the delta CT method. I want to know if there is something I am missing. Can you please comment on the 48-well plate layout? I would appreciate your input.
looks good!
What instrument are you using?
I use an ABI 7900HT and for that instrument I find it useful to put my duplicates in rows not columns. There is one graph that is built in that shows CTs vs wells. It is an easy way to see if your replicates match. The way that graph is laid out, it put wells next to each other that are in a row. It's a minor point really. In the end, they layout isn't so crucial. You will get your data off the instrument and put it in excel probably.
Thanks. I am using step-one qPCR system. I am not sure about it so I will contact tech service tomorrow. Also some people suggested that I need to do a standard curve. However, to my understanding standard curve is not needed for relative quantification. Do you think I need to perform a standard curve?
Thank you! I am actually using SYBR Green so do you think the same dilution can be used?
A general comment:
Often, the temperature homogeneity in the block is not perfect. Therfore, there should actually be no systematic pattern of the samples or conditions in the layout. However, a randomized layout (or some other more complex layout with a better distribution of conditions and replicates) is difficult to pipet and more error-prone. One has to consider this trade-off.
If you can, leve the corner positions empty, since they are mosl likely and most severely affected by temperature deviations. If this is alos possible, leave the border positions empty as well (only if you don't need the wells anyway).
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