Does anyone have suggestions, beyond using the correct size tips, for pipetting small volumes (2 uL) of high concentration (+400 ng/uL -2000ng/uL) DNA?
Are these in tubes or plates? What kind of medium are they in, and what platform are you using or looking to use?
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Plates, Armadillo 96 well PCR, 15 uL in well, water and I am using a Bravo with no liquid level detection.
I’ll also be dispensing into ~ 100 ul of water and mixing.
What’s the final conc? Is an intermediate dilution a possibility?
The Bravo should be able to easily do what you’re asking. Are you trying to cherry pick samples? Or just straight stamps of 96-well plates?
Edit: actually, I assumed you have the 96ST head on the Bravo. If you have a 96LT then yeah, 2uL is a bit low to be comfortably dispensing. What head do you have?
I have a 96 ST head (and a couple of others as well) but I am using the ST head.
It’s a stamp.
This IS the intermediate dilution. It should be more precise than a person doing it.
We are doing high concentration DNA Quants and need to do a 1:50 dilution. We cannot just add water to dilute because we cannot guarantee a user would pipette as accurate an amount. (I can accutally show through data that most of them cannot.)
I think I now have the method validated however. I am working with a fluorescent measurement kit so that is roughly 5% error right there but I’m at about a 6% error rate.
I just wanted to know what people’s favorite ideas were. I do not like reverse pipetting for this. It was neither precise, nor accurate.
The kit itself has some special considerations such as light and temperature sensitivity and the only way to actually validate it is to utilize a known, validated, lambda DNA sample or something similar. I have to validate the control daily on a nanodrop, so I need to have a very clean sample. Lambda works well for this.
When I had to do a 2uL xfer with a 1200 uL 96-head, I just followed the basic advice and performed some troubleshooting for final tuning - sounds like you had the same experience.
- Enable a larger blowout airgap, before liquid aspiration to counteract pressure.
- Aspirate medium-slow, without liquid following and a decent-but-not-excessive submerge depth.
- Reduce Z-travel speeds to 5 or 10% of max to prevent jostling.
- Prime the dispense by over aspirating then dispensing back to source 1 or 2 times, if possible, and dispense into liquid either right at, or right above liquid surface.
- An additional “tip dip” at dispense can also help overcome tension and backpressure in the tip if the small volume is proving to be difficult to dispense from the tip.
- If this is a single disp, add a post-mix and blowout
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We do something very similar. We have Hamiltons and regularly pipette 2ul of DNA into 18-98ul of TE for dilution. Our validations included DNA as high as 500ng/ul and our CV’s are always very tight.
-We had our Apps Scientist come in for the day to really dial in the liquid class (shout out to Chris at Hamilton). This made a big difference.
-Use 50ul tips. For the Hamilton, the are able to pipette reliably down to 1ul.
-Prime the tips by “mixing” 2-3 times before pipetting 2ul. Add a mix after aspiration also.
-There is an Artel poster floating around that shows removing the pipette tips from the liquid after priming, but before you perform the actual aspiration step will get you a better aspiration. I’ll try to find it.
-Add a larger blowout (try 5ul). Dispensing 2ul is quite a small volume and the liquid often gets stuck in the tip.
-Surface dispense, 1mm into the liquid surface. Sometimes the 'dispense at bottom" doesn’t work well for this small volume whereas the surface dispense consistently pipettes 2ul.
-MIX THE DNA AND DILUENT BY PIPETTING. You may not really have an issue if you are using the shaker to mix. We first thought that we had a pipetting issue because we were mixing with a shaker. Turns out the shaker can not thoroughly mix small volumes in a 96 well plate. When we switched to pipette mixing we had consistent results.
Hope this helps!
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