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7500 Reaction Volume

qPCR troubleshooting

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3 replies to this topic

#1 4N6Addict

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Posted 10 May 2012 - 05:44 AM

Does anyone know if there is a way to change the reaction volume of a run in the 7500 Real Time system after the run has completed?

If not....any ideas on how to proceed, such as normalizing my data?

I would just rerun everything, but my samples tend to degrade pretty fast so I know the results would not be accurate.

Thanks!

#2 leelee

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Posted 10 May 2012 - 08:49 AM

I don't think you can. I thought that the volume was used to help determine the ramping times for each temp cycle of the reaction. So I think its too late, and the run would have been optimised for whatever volume you entered.

Whether this is a major problem or not I'm not sure. What was your volume and what did you set it at?

As for normalising your data, sorry I'm absolutely no help to you there...sorry. Hope someone else has some ideas!
(maybe comparing your standards to another run?)

#3 4N6Addict

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Posted 11 May 2012 - 07:45 AM

I don't think you can. I thought that the volume was used to help determine the ramping times for each temp cycle of the reaction. So I think its too late, and the run would have been optimised for whatever volume you entered.

Whether this is a major problem or not I'm not sure. What was your volume and what did you set it at?

As for normalising your data, sorry I'm absolutely no help to you there...sorry. Hope someone else has some ideas!
(maybe comparing your standards to another run?)


Thanks for the reply! That's what I was afraid of...My volume was 15uL, and I forgot to change the default before my run, which was 50uL Posted Image . I went ahead and re-ran everything, but like I said I'm a bit worried about the stability of the DNA so I know these results do not accurately reflect what I would have gotten right after extracting.

#4 bigudukaz

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Posted 27 March 2013 - 03:28 AM

Well if You set bigger volume then the one you added - the ramp speed will be slower (machine thinks that it takes 1 second longer for the sample to reach the temperature).
I was using StepOne plus and 7500 set to 100ul sample volume, when i had only 10ul. There was no difference in results compared to the ones where the volume was set correctly.

The problem that can come up is when the polimerase cant finish folding and copying the fragment before the next denaturation.

Hope that helps





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