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Reducing Pipetting Error? - How to improve... (Jun/13/2010 )

Hi guys,

I've been working in a lab for about 2 years now, and over that time I've noticed that my pipetting skills have improved quite a bit since I started. However, I'm still not 100% happy with them. My western's turn out fine, but sometimes when doing qRTPCR (with small sample volumes, like 2.5ÁL of cDNA and 22.5ÁL of mastermix), I get rather large standard deviations. Similarly, when doing a BCA assay to measure protein concentration, I always get at least one sample with a %CV of 7 or so (all the others are lower, usually 0.2 to 5%).

I know that SOME error is inevitable, but I'm trying to minimize it as much as possible. Here are the things I'm doing to make sure that I pipet accurately (if it's important that things be very precise):

-"Pre-pipet" the liquid once before delivering the volume

-Making sure to hold the tip level with the solution

-Vortexing the solution before addition

-Making sure not to insert the tip at different depths each time (so that the sides of the tip don't get coated with liquid)

-Minimizing how much the sides of the tip come in contact with the solution that I'm taking from or adding to (to eliminate extra volume delivery)

-Relaxing my grip on the pipet between pipetting to ensure that it does not heat up too much and change the volume of the air gap (also wearing gloves of course)

Is there ANYTHING that you guys have found helpful other than the things I listed? I know that I will never be "perfect," but I'd like to think that I can get "errror-free" (all CV% under 5%) for a BCA assay or something.

One other thing is that we use manual pipettes (not digital/automatic) so _I_ have to improve -- I can't buy new pipettes! The pipettes I use were calibrated 2 months ago, so they should be reasonably accurate. Also, they are nice quality (eppendorfs).

Any advice is appreciated!!

-ThePipetMan-

1. Always prepare master mix whenever possible
2. Use low retention pipette tip
3. Avoid pipetting vol less than 1 ul. If possible, pipetting vol > 2 ul
4. Will not use certain pipette brand (but won't mention it here) due to difficulty in securing the tip tightly to the pipette "nozzle"
5. Will check tip after dispensing to ensure no residual liquid got stuck in the tip (if not using low retention tip)

-lsek-

If there are outliers once and a while you have to practice again and again, you have seen your skills improve over the last 2 years and it will get better intime although it will go slower and slower.
Be concentrated and let nothing distract you when you're pipetting. Try to get in a constant flow to get every thing as stable as possible as the speed of pressing the plunjer, the waiting time for the liquid to flow downwards before you press the plunjer completely down etc.

-Gerard-

You can also read this:
http://www.pipettecalibration.net/pipette-...Pipetting-2.pdf
Full of good information that can be extended beyond that particular brand of pipettes.


But I would add that for your qPCR, preparing large batch of master mix is probably the best thing you can do, as it will considerably reduce the errors inherent to pipetting small volumes.

-Canalon-