qPCR issues

[anna_m]

Hello, we use Kapa qPCR kits to quantify our libraries before sequencing. Recently, the NTC wells are giving low cq values which means there are contaminants and therefore the data is not reliable. Despite cleaning the wells, changing kits, and re-aliquoting reagents, the NTC wells are still giving the same result.

Has anyone else had this issue? Does anyone have any suggestions?

Thanks in advance.

[microgirl123]

How low are your values? Occasionally my NTC wells come up with very, very low values (way below the final standard). I just ignore it - compared to the inaccuracies caused by dilution and pipetting, it's meaningless.

[anna_m]

We have a minimum cut off of 28 for the cq values and we have been getting as low as 18 recently.

[helskel]

What do you mean by "cleaned the wells"? Do you re-use plates? That could be a source. Also, how clean is the water that you use for dilutions? If you didn't get new water for your tests, that is also a possibility. What do your NTC wells contain? Do you just use prepared Master Mix, or do you also add water?

[anna_m]

No, we don't re-use plates. We clean the plate-holder wells with bleach, water and ethanol on recommendation by Kapa once a month. We don't use water to dilute the libraries, we use EB-tween. We put master mix and EB-tween in the NTC wells.

[Jessica_L]

Other than a few incidents, my experience has been the same as microgirl123-- if my last standard has a Cq value of 35, I might see some signal in the NTC around cycle 38, which corresponds to such a low level of DNA contamination I basically just don't pay it any mind.

When I've had issues in the past with higher levels of contamination in NTCs I took a pretty extreme approach-- use a PCR enclosure, wipe everything down with bleach then ethanol, including the enclosure surfaces and pipettors. I'd also usually leave the UV light on in the enclosure for at least thirty minutes prior to setup and made a point to leave pipettors, tips, water, any other non-sensitive consumables (i.e. plates, plate seals) in the hood.

I've also noticed that not all plate seals form a good seal against the plate, which would occasionally cause me problems when the plate was full enough to have NTC wells near my unknowns.