Hello,

I'm just coming to the end of my first year of my lab based PhD. Before entering into this PhD I hadn't had any experience of working in a lab as my undergrad. dissertation didn't require it and I did an anatomy based degree with no molecular biology practicals. Anyhow, it has been a steep learning curve to integrate into the group, get my head around the topic and learning lab skills. I really struggle with lab skills, my supervisor didn't seem to think it would be a probelem me not having done lab work before but I'm now having problems. Specifically my pipetting is rubbish, I'm running assays and it's not good, I've had someone check my technique and have done a little practise with water on the recommendation of my supervisor (very boring). The second scientist in my lab informed me that if I was working in the NHS then my assays wouldn't be good enough and wouldn't be used. I'm supposed to do the assays for the whole lab and am getting worse if not better, I'm sure they were better earlier on in the year! If anyone has any advice it will be greatfully recieved!

it comes with practice, lots and lots of practice. it takes time sometimes to "hone" the skills.

when you say your pipetting is rubbish, what kind of volumes are we talking here?

1000ul and less?

advice given to me, when i first started pipetting, was to take up the volume slowly. check with the eye that there were no bubbles. and then slowly depress the button and pippete down and then at the end doing the extra push.
lab skills, it's just a matter of practice. don't worry. you will get there!

Thanks for the encouragement. We do a pipetting exercise with left over radioactive solution - pipetting 10 samples of 100ul, 10ul and 1ul then running them through the gamma counter, we then look at the error between the samples. I've been told I need to get it down to an error of under 5% for all of them, last time it was 14.3% for the 100ul, 15% for the 10ul and 25.3% for the 1ul. The crucial amount for my assay is 200ul so in theory I should be able to do it!!

you will do it

try maybe touching the side of the well when you pipette out, so that the last remaining liquid is taken out due to the cohesion property of liquid.

Like laura said it comes with practice. Some other tips, which may or may not be useful.

If I'm doing a lot of pipetting in one sitting I make sure my arm is stabilised. either on bench or against my body.

If you have trouble with getting the tip to the well accurately use the index finger of your other hand to stabilise the tip of the gilson (not touching the actual tip though, just above the tip), this cuts out any shakes in the hand.

When I'm pipetting small volumes into or out of eppendorfs I always hold it up to your eye level so you can see the liquid drawn up, and expelled.

Don't draw liquid up to quickly or it creates bubbles in the tip and draws up an inaccurate amount.

Generally just work on your technique, and try to keep it consistent.

Like Laura said You'll get the hang of it :O)

Is there any way you could get hold of positive displacement pipettes? They're great - any air bubbles are obvious to spot so accuracy is excellent. I use them for ELISAs which have to be accurate/precise within 10%.

This may sound obvious but have your pipettes been calibrated recently?

I spent a month doing PCR recently: I wasn't used to pipetting such small amounts and it took ages to get the hang of it (and I've been doing lab work for years). Luckily I was given friendly encouragment, instead of being told - like you were - that the assays were not good enough. I got results after a LOT of attempts.

The second scientist sounds like a right miso to me. Surely he/she was new to this once?

The callibration question is a really good point - especially if your value are always out in the same direction (e.g. always too high).

Try using a different set of pipettes, but also do ask about callibration.

Hi Walrus,

I regularly run assays with volumes of 2ul of less so have been through exactly what you are describing! I come from an NHS background but the area I worked in previously involved the use of relatively large volumes (200ul +). I have found the main factors in getting your % CV down for small volumes are;

(1) Ensure the tips you are using are v. good alternatives (if Gilson brand are not being used).

(2) Pipette onto the side of the eppendorf/well so that you ensure you get the full volume - this also helps to prevent aerosol.

(3) Centrifuge all solutions if possible prior to pipetting as this helps disperse air bubbles somewhat. Obviously this does not apply if you’re working from stock bottles!!!!

(4) Calibration! I think with Gilsons you should pipette a volume of water at the lowest and highest volumes possible (5 replicates) and take the mass on a 5 digit balance. If they are really bad (some times there is a slope i.e. they are higher at the lower volumes and lower at the higher if that makes sense! - I would send them off for a full service in this instance).

Do you always do your assay in a room with a constant temperature? When pipetting very small volumes differences in temperature can change liquid volumes thereby increasing you error %. I know this only makes a small difference but sometimes lots of little things happening together can lead to large % errors.

Don't worry it will come then you'll be a pipetting master!