Unequal amplification efficiency of heat blocks - (Apr/20/2005 )

hi all

I used thermal cycler with two heat blocks, one for 0.2 ml eppendorf the other for 0,5 ml. I make a master mix then divide it to tow tubes (0.5 and 0.2 ml). I run PCR in my thermal cycler, two block parallel with the same thermal cycler.
But only samples from tubes 0.5 are positive. What's problem here? Please help me (DNA templates are the same for all samples).
Thanks.

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hi there,
well either the tempr is not evenly spread throughout the thermocycler...it happens, once i had read an article...regarding the effect of placing the pcr tubes in a thermocycler in different places and their relation to amplicons

or it must be that u must have forgotten adding some ingredient in to the mix like primer or template......

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Hi biodragon123

This could be due to a couple of things, the first is the thickness of the tubes, is the thermal cycler set up for calculation of different size tubes. Remember in the 0.2ml tube you will have a larger surface area to volume ratio and therefore would ramp more quickly between temperatures etc.
The second possibility comes from this as well, template can potentially stick to the side of the tubes and won't be effectively amplified, again the small tube has a larger surface area to volume ratio and therefore more template may stick to the tubes. This can potentially be overcome by addition of a small amount of BSA.

Hope this helps

Scott

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Thanks for your replly.

Ok, when I run PCR for sequencing, I use some tubes and put it in different places but in this case it isn't problem.



Oh, no, I make master mix and devide divide it to two tubes (0.5 and 0.2 ml), in 0,5 ml tubes have amplified product.



This is problem which I was unthinkable. Thanks

Do you think that one heat block (for 0,2ml tubes) in my thermal cycler was broken down.

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I suspect the problem is not that the heat block is broken down, as presumably you are still doing other cycling reactions on it ie. sequencing etc.

Just that you may have to adjust the parameters a little when you are scaling down into different tubes. If your temperature is calculated by block temperature then the sample is going to be hotter for longer in small tubes, if it is calculated by sample temperature remember to change the setting for the different tube sizes. However, I still think that something like a little BSA may help your cause.

However, if you have the PCR working in 0.5ml tubes I would just use those tubes - don't change things that are working.

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