Over the last two years, I’ve run over 30,000 qPCR reactions to verify transcription factor targets using chromatin immunoprecipitation. Because of the large number of reactions and the expense of qPCR master mix, I run these qPCR reactions with low-volumes (10 ul total) in 384-well plates on an ABI 7900HT qPCR machine. I’ve learned to tolerate the tedium of filling each of these plates, but I’ve never learned to tolerate my low-volume multichannel pipettor (i.e. 0.5 ul - 10 ul). I fill the plates with 5.5 ul of master mix, 3 ul of template, and 1.5 ul of primer, so I really get a feel for how the pipettor operates across a wide-range. And until a few weeks ago, my opinion was that all low-volume pipettors are mediocre at best.
Every multichannel pipettor with an operating range of 0.5 ul - 10 ul has about the same accuracy (typically +/- one or two percent). This performance is measure by the pipette calibration folks who very carefully make sure that each tip is evenly sealed onto each channel and slowly aspirate each sample. They do this with a number of samples and use a very sensitive scale to judge (and adjust) the pipettor’s accuracy.
For the larger volume multichannel pipettors (>10 ul), I think this is a pretty decent way to judge a pipettor. But for the low volume multichannel pipettors, I find that in practice the experimental pipetting error does not correlate with the accuracy of the pipettor in this ideal calibration scenario. The problem is that when you are filling plates and you don’t have all the time in the world or the muscles of a professional boxer, the error from poor tip sealing is much greater than that from poor calibration. After trying a couple different models from different companies, I had resigned myself to the belief that it is impossible to have a small volume multichannel pipettor that easily, evenly, and consistently seals all of its tips.
Since life must go on even in an imperfect world that lacks good low volume multichannel pipettors, I developed a number strategies to improve the consistency.
First, every three to four 384-well plates I would apply a very thin layer of silicon grease to each channel and carefully wipe it off (like waxing a car). This drastically improves the seal of the tip. However, the pipette calibration folks told me this isn’t terribly good for the pipettor, as if you aren’t careful and you glob the grease on, some can get aspirated into the channel and gum everything up.
Second, I use only the tips recommended by the pipette’s manufacturer, and I apply very firm pressure to the center, left, and right of the pipettor to seal the tips (but I DO NOT bang the pipettor on the tips; banging the pipettor on the tips can easily break, crack, or warp a channel). The problem with this tight seal is it really wears you out (both to seal the tips and to eject them). By the end of a plate or two, I sometimes break a sweat. But come on this is a lab not a track, I’m not supposed to be sweating.
Finally (and most importantly), I use my peripheral vision to quickly check each channel by eye to make sure they have roughly the same volume. If they do not, I eject the tips, firmly attach a new row of tips, and try again. This visual scan is essential to prevent having a handful of qPCR reactions that fail because some of the tips didn’t aspirate any liquid sometimes.
When my workhorse low volume multichannel pipettor recently broke after many years of use (Finnpipette 12-channel; Part No: 4510010), I looked around for something different. Most manufacturers claim their pipettor has a great tip-sealing mechanism, so you can’t really go by that. However, the new Biohit mLine and eLine pipettors have what they call the Optiload tip loading mechanism. With the Optiload system, the channels are spring loaded. Biohit doesn’t provide much information about how it works or why it works, they just say it’s wonderful. But if you think about it, when you load a standard 12 channel low volume pipettor, you are pushing 12 fixed circular pieces of plastic (the channels) about 2 mm or so into 12 slightly larger circular pieces of plastic. Since everything is fixed, if one or two tips (or channels) isn’t perfectly even with the others, that tip will either not seal or it won’t seal evenly. I’m sure if you’ve used multichannel pipettor, you’ve lifted your pipettor a few times where not all of the tips were attached… So if things don’t line up perfectly, you have to push harder to bend the tips or the channels slightly to get everything to seal properly. This pushing places a load on your arms and an additional strain on your wrist when your try to eject the tips that, through your firm pushing, have become almost a permanent part of your pipettor.
So I bought the mLine mechanical pipettor from Biohit (12-channel model m10; Part No: 725220; I bought it from Fisher) hoping that these springs would allow the channels to adapt and more easily form complete seals despite the unevenness of the tips in the tip tray. Given my past experience with low-volume multichannels, my hopes weren’t very high. However, after running over 1500 qPCR reactions with this pipettor, it has become one of my prized lab possessions. The tip sealing is superb - none of the other low volume pipettors I’ve tried even come close.
Although, in the pictures on Biohit’s website there appears to be a spring on every channel, I really can’t feel the springs in the center. If you press hard on the right or left of the pipettor as you are attaching the tips, you definitely notice them. I find I get the best tip seal if I apply moderate to light pressure in the center followed by a slight pressure on the left and right; it’s almost as if the springs allow you to roll the channels across the tips - evenly sealing them one-at-a-time.
Much of the mLine pipettor brochure from Biohit was devoted to how the mLine pipettor lowers the risk of Repetitive Stress Injury (RSI). I bought the pipettor solely with the hope that the tips would seal evenly. However after using it a good bit, I find I appreciate how this tool is easy on the wrist. First, you don’t have to press very firmly to get a good tip seal. Second, because the tips aren’t jammed onto the channels very hard, the tip ejection is spectacularly easy. According to Biohit, their piston is much easier to move up and down. I agree, but in some ways that makes the pipettor feel a little wimpy to me (though I guess my wrist appreciates it). And finally, the pipettor is extremely light.
With the spring loaded channels, it takes a little practice to get a feel for how much pressure is necessary to fix the tips on. I noticed I get a nice even seal even when I barely press at all. However, even though the seal is good, the tips sometimes fall off if you don’t press hard enough (in many ways it is remarkable how good their system works when a barely attached tip is still sealed even enough to pipette accurately). However, with much less pressure than a typical low-volume multichannel pipettor, you’ll be able to evenly seal the tips, and they won’t fall off.
A second quirk is that there is a little blue button on the pipettor shaft. You must press this button down while you adjust the pipetting volume. I didn’t like this feature at first, but now that I’m used to it, it’s no big deal. We put a note on the one in our lab, so that people know what the button is for.
I’ve only had the pipettor a month or so now. When I purchased it, I hesitated a little, because with all of those extra springs in the pipettor, I was afraid it would be more likely to break. So far the pipettor seems pretty tough - time will tell. If it lasts more than a year or two, I’d be hard-pressed not to buy another one.