Pipetting guidelines in qPCR

[ecoor1]

Hi,

so I'm about to start this new project dealing with DNA copy numbers. I just read through the kit Quick reference card (http://tools.invitrogen.com/content/sfs ... 062367.pdf). It says on page two, at section 4: "Mix the reaction mixture with the gDNA by pipetting up and down several time". I've done lots of qPCR but never did this occur to me. Is this really needed as you might think the stuff gets mixed once you centrifuge it?

E

[and3k]

Hey,

for my qPCR assays it is needed. I tested standard dilution series with and without mixing by pipetting up and down, and the quality of the standard dilution series decreases without mixing.

Best,
Bela

[relaxin]

I agree. I normally pipet up and down a few times and stir up the solution with the same pipet tip. The heating may help mixing the solution, but optimal amplification may be delayed a few cycles. For qPCR, this delay will affect the CT values.

[mchlbrmn]

If anything I think spinning would segregate the sample more.
I believe I read originally that convection from the heating/cooling was sufficeint to mix the sample. I added on one up and one down to my pipetting at one point, but my dilution curves come out well either way. I find it very annoying when my pipetter sometimes gets some liquid stuck up high in the tip that I can't expel easily after I pipet up and down too much. Now that I use a multichannel, this is even more of an issue.
My reagents viscosity and etc. could be different from yours, so you might run an extra dilution series to test this.
When I make the serial dilution series, I always thoroughly mix and spin the dilutions, before I take some to the next dilution with a fresh tip.

[greencells]

Mixing is probably necessary because of the viscosity difference between the reagent mix and the DNA sample. I don't pipet up and down -- too sloppy, possibility of losing sample -- rather I invert the plate several times after capping/sealing the wells, then spin briefly. Based on repeatability and sample-to-sample consistency, this seems to work fine.

[relaxin]

That is a good idea. But I think the 10 ul of solution may stay at the tip of the wells. You may have to hit the plate on a stack of paper towels a few times to get the solution inside mixed.

[selester]

While unorthodox, and contrary to conventional wisdom, I solve the mixing problem by adding the DNA first, followed by the reaction mix.
The key is to add the DNA carefully to the bottom of the PCR well.
This has the added advantage that, if you get interrupted or muddled when adding the DNA, you can see where you are up to!
I have used this technique for several of these Life Technologies qPCR gene copy number assays - which seem to work very well indeed.
I modified the reaction slightly by adding 2uL of DNA (at 10ng/uL) and 18uL of reaction mix.

[LeserattePD]

When you add your gDNA to the PCR reaction mix, the volume of gDNA is generally much lower than the PCR reaction volume.
Also, when you pipet, you loose a certain percentage of your liquid sticking to the tip (let's say 5%).

Therefore, if you do NOT pipette up and down, you loose a certain percentage of the gDNA volume within the tip (5% of your gDNA - I use 4 ul). Let's assume there was 100 ng of gDNA per 4 ul, then you've just lost 5 ng of template.

(BTW you should never pipette less than 2 ul of gDNA anyway to achieve a high accuracy, dilute with H2O if neccessary)

If you do pipette up and down, you loose the same volume of liquid on the tip (since it is the tip/volume you used to pipette your gDNA with), but your gDNA is diluted with the PCR reaction (in my case 16 ul), thus the actual loss of gDNA on the tip is much lower (1ng as it is a 1:5 dilution).

Pipetting up and down is a standard pipetting technique that should be used whenever you are pipetting, not just to mix your reaction, but to reduce the loss of (undiluted) liquid that you are adding to another liquid. A quick vortex and then spinning down is another of those standard procedures that should be used when setting up reactions, except when pipetting materials that are sensitive to mechanical disruption (such as DNase or competent bacteria while transforming).

I hope this helps.

[ecoor1]

Thanks for all the tips!

Previously, when I didnt do any up-and-down pipetting, my Ct-values were highly consistent; ~standard deviations always below 0.1. Now that I tried this "new" technique, the SD's were huge and there were bubbles present in every single well. Maybe it's because of the small reaction volume (20µL) and lack of practice.

Thanks,

Tapsa