Use of ROX in qPCR - (Oct/06/2005 )

Hi

I use Invitrogens Platinum SYBR green qPCR supermix and have had no problems with it so far. I don't add ROX into the master-mix: I tried a couple of plates with half ROX'ed and half not (duplicate sample sets) and didnt notice a difference. Does anyone have any views on the matter? Some people in my lab just add the ROX in anyway and I'm wondering if I should do the same.

Thanks

Auriol

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ROX is a control; you never miss it until there are problems that need to be corrected and you're ripping your hair out trying to find the source

ROX, being the passive reference, normalizes your replicates against one another. This is very important if you get a little air bubble in your pipettor tip one day and end up with 24ul instead of 25 ul in your PCR well...the ROX allows the software to adjust for minute differences in amounts added to each tube.

I think the major difference (assuming you're not troubleshooting a problem) is that your replicates will be tighter if you use ROX. Anything to give you better statistics and more reliable replication of the experiment should be added, is my opinion. Compared to the other reagents and the time you take, the ROX is cheap insurance.

Up to you...but I think it is important.

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Some of the machines from ABI require ROX in the reaction. I think it was origionally intended to force people to buy their reagents as well as an internal control.

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Hi

Thanks, probably is better if I use it I suppose. Better to be safe than sorry.

We have Stratagene Mx3000P machines and they allows for either with or without ROX setup. We also have ABI but I have steered clear as other people have mentioned that the setup can be troublesome.

Auriol

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I use the Mx3000P as well. I called Stratagene about using ROX and they recommended it to especially correct for differences in the thickness of the plastic tube caps. This would interfere with the readings I'm sure.

-Hank

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Does anyone know what the typical concentration of ROX is per reaction? I've just got a qPCR machine and don't want to be tied in to using commercial master mixes so would prefer to make my own.

Thanks a lot

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I can check out the ROX concentration that I used when I get to lab later today.

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Dear DanT,
I always use ROX in final concentration 60nM per reaction. This is acroding to Synthegen recomendation.

Best regards

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Actually, the product manual of Stratagene's Brilliant SYBR Green Master Mix recommands different concentrations of Rox on different Real-Time system. You should really check what's recommended for the machine you are using. As far as I remember, the recommended amount for Stratagene machines are different from ABI or Bio-Rad.

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