Dear Noah,

I don't use 96 well plate for PCR (closed system Lightcycler, capilaries version; but there is also a LC version for plates).

So to precise, could you describe me the process for pipetting the different reagents and delivering to the plates : multi-canal pipette ? under laminar flow ? if multi canal, with (x) filters cones ? Is it an automatic pipetter ?

For the qPCR step with the 96-well plate: is it strictly closed ? by a part of the machine ?

Thanks

Best regards

Didier

Hi Didier,

Thanks for taking the time to reply.

We make our supermix in a PCR hood (everything except the template).  In a secondary hood we add our template to individual wells in the 96 well plate. We use a single channel variables volume manual pipette with barrier tips. So the template isn't added until we are in a dedicated PCR room in a dedicated template PCR hood near the cycler.

The plate is completely open as the seal is a single piece that must be placed on after the wells are filed. We do have an option of using strips of 8 or 12 PCR tubes, but this increases our cost and decreases our throughput.

It is a hot-start machine so we don't use mineral oil to prevent evaporation, it is a completely closed system.

I hope this answers your questions, and I look forward to any feedback you might have.

Kindly,

Noah

OK NoaH,

I understand your system.

So, I think you are right in your interpretation showing that with positive well in the center of plate, you have contamination, and not if it is in an external well.

When several tubes or wells are manipulated simultaneously, that's better (with individal tubes) to open and close each time a tube one by one when delivering reagents.

That's not possible with a 96 well plate (too much time); and in general, it is at this step that you can have inter-tube (or well) contamination, but also mix tube contamination  if it is open; because you have always microaerosolization of liquid that can go down at random in the nearest tubes; especially because most of laminar flows (when you use it) are with vertical stream.

But if you think that it can be the case (argument with your experiment positioning differently your positive control), that's not in favour of an external contaminant, with the restriction: if all other wells are contaminated, not only some, in general the meaning is that it's the tube with the common mix which would have been contaminated,

In the first case, that's is simple to solve, as you done: use only wells distant from positive controls and isolate this one from other, even in a same plate.

More, in a general way: when it's possible, that's better to manipulate under laminar flows, with one underwhere you prepare the common mix, another to deliver the mix in each well with extracted DNA, and a third zone where you have your amplfied product manipulations (I suppose that the revelation system can be variable).The more contaminated area is the PCR room because 10exp6 concentrations of DNA is produced, and these area has always contaminated atmosphere after 2 or 3 years of use ; especially if systems of revelation (UV gel detection, flurorescence ...) are open

If you have more questions ?

Best regards

Didier.

Thanks Didier for your input on this.

Hi Noah, I only want to give some other advices.

I don't know if you used a support for your 96 plate, even if you clean well your PCR hood, it is better not to put directly the plate on the bench or on ice, those are sources of contamination for example.

In my case another source of contamination was the machine itself, I have in common the 7500 Real-Time PCR System - Life Technologies with other users, so I have to check the machine block contamination. Sometimes the contaminated well, can give signal also in NTC samples.