You should decide on the "steps" of you dilution series depending on the anticipated range of copies in your gene of interest. Most folks will use a 1:10 dilution series or 1:5. If you have a very narrow range and need more precision, you can try 1:2. I'd start out with 1:10, don't go lower than a calculated 10 copies/reaction, and see what you get.

Good luck!

Katie!

Many thanks for your quick response. I'll first try out the 1:10 as suggested.

Regards,

Geoffrey

I suggest you make serial dilutions of a factor of 10 say from 1:10, 1: 100, 1:10^3, 1:10^4, 1:10^5, 1:10^6. Run this and determine an optimum concentration to use corresponding to a Cq that is not very high. Values between 10-20 correspond to high copy numbers. You can work backwards from the Cq value to calculate the no. of gene copies corresponding to a particular Cq value. Aim to use concentrations that are not to high and and not too low. A concentration that gives a Cq value of between 20-30 will be good.

As far as dilutions are concerned, good answers were given. Now if you want to compare your PCR machines, you need to perform the exact same experiment on both devices at the same time. I would recommend to set up the exact same PCR plate in duplicate and run it on your two machines. Then you can see which one gives the better efficiency, the better slope... have a careful look for the very diluted samples, beacuse it's usually the ones which are not very well amplified and which affect the efficiency curve. Good luck!

I recommend to make serial dilutions from 1:10, 1: 100, 1:10^3, . Run this and determine an optimum concentration to use corresponding to a Cq that is not very high. I would recommend to set up the exact same PCR plate in triplicate and run it on your two machines. Then you can see which one gives the better efficiency, the better slope. A concentration that gives a Cq value of between

20-30 will be good.