The Standard Curve shows critical serial dilution points used to prepare samples for Next Generation DNA Sequencing. Technologists with manual pipettors are still often trusted more than automated systems when the highest accuracy is demanded for tiny and submicroliter volumes. Hal Schneider of Claritas Genomics in Cambridge, Mass gave an excellent presentation of why it really is so important that the best possible precision and accuracy are achieved in the critical early dilutions. His presentation can be seen by clicking on the graph or here.
APPLICATION 1 Next Generation Sequencing (NGS)
The Tufts University School of Medicine, Department of Integrative Physiology and Pathobiology, uses the DRD. This is the University Core Facility.
“We find it easy to use and very reliable as well as very accurate at low volumes. The contact-free dispensing is a nice feature”.
They use the DRD Differential Pipettor to deliver 0.5 uL (500 nanoliters) of NaOH contact-free to prepare DNA samples for NGS in the Illumina.This lab tend to follow the Illumina protocols but modifed them to use only 0.5 µL of the NaOH.
The Differential Pipettor, dispensing by its contact-free Blastoff capability, is free from any “wickoff” or other touchoff dispensing related errors. The data shows that dipping the tip during aspiration more than the correct 1-2 mm results in actual increased aspiration from gravity/hydrostatic effects.
Measuring Tiny Volumes Dispensed By Our Differential Pipettor
This is shown first by preferred contact-free “Blastoff” and then by conventional touchoff-and-drag.
Contact-free dispensing to the Artel PCS vial is done cleanly from a convenient height. Touchoff dispensing is done in the standard manner by touching the tip to the inside wall and dragging it while dispensing.
The movie shows how nice it is to dispense by contact-free Blastoff. The tip does not need to be placed at any precise distance or angle, you don’t need to pause or navigate the tip, and the tip is clean and free of residual at the end.
More Accurate Low Volume Pipetting Really Does Facilitate NGS
Differential Pipetting facilitates NGS, aka “Yes, Virginia, accuracy is important” and “Pipetting 101 for NGS Dummies”. Next Generation Sequencing people will recognize the curves on the whiteboard in the background. This is a nice introduction to the importance of accurate pipetting at the outset of the process.
At about 1 uL, dispensing by conventional touchoff-and-drag is here adding approximately 2-3% extra volume from “wickoff” of volume on the outside of the tip. The Differential Pipettor provides data to show this because it can dispense both by the conventional touchoff-and-drag technique and by contact-free “Blastoff.” Shown for correct shallow tip dipping during aspiration and for deep dipping.
On a civilized tarmac in the hallowed halls of the Ploegh Lab at the Whitehead Institute in Cambridge, Massachusetts, Gilson PipetmanP20 – the great French 20 uL champion – and Differential Pipettor DP20 – the differential displacement, dual resolution, contact-free dispensing challenger from Gloucester, Mass – are both standing ….after what was supposed to have been an uneventful charity event.
Precision and Accuracy has been declared a STATISTICAL DRAW by the exceptional judges. But the crowd is roaring! There was something different about this challenger. And later, in the taverns and the think tanks, in the dusky machine shops where the coffee is not cappuccino and in the shiny glass-and-light festooned edifices of exceptionalism, de Tocqueville’s revered roots reigned. They started to figure it out.
An experienced pipetting person using a top-of-the-line pipettor and tips with which he was very familiar got fully comparable precision and accuracy with a new Differential Pipettor that he had never seen before – delivering contact-free, untainted by touch or immersion! But the crowd was already on its feet demanding a re-match for a fuller look at accuracy, range, speed, and ease.
Ella used a 20uL Differential Pipettor to transfer approximately 20uL of hydrochloric acid in a toxicology application in which precision and accuracy happen to be irrelevant. So why would she have used the World’s first Differential Pipettor, uniquely able to pipette down to 250 nanoliters and deliver contact-free with unsurpassed precision and accuracy? Read the article for this interesting little story. The World’s first Differential Pipettor is turning out to be the World’s coolest hand pipettor – period!
Differential Pipettor Matches Gilson and Rainin Best From 10 µL Through 0.75 µL
Theatrical version
On January 23, 2015, in the NIST-tethered-and-trusted high-tech Calibration Laboratory at Artel, in the fine hamlet of Westbrook in the great state of Maine, the venerable French and German champions Pipetman P10 and Rainin 2µL squared off together against four new differential displacement, dual resolution, contact-free dispensing colonial challengers from Gloucester, Massachusetts. A mere 2 days earlier, on January 21, 2015, a 20µL Differential Pipettor had stood against Pipetman P20 in the hallowed halls of the Whitehead Institute in Cambridge, but that crowd so roared its demand for Truth down to the lowest levels that only divine intervention could have called forth today’s contest so swiftly.
Different today is that the Differential Pipettor’s manager, Dr. Donald Schwartz, was not present to provide even moral support let alone observe, and was in fact so unaware of the contest this day that no communication on any level would have been possible. In contrast, managing and manning the French and German champions against the spiritually denuded differential pipettors was none other than the 6′ 6″ rail-splitting head of the Artel Calibration Laboratory, a Lincolnesque figure (in both stature and scientific integrity) whose pipetting skills with the reference systems he used unquestionably strode far above excellence in the realm of true expertise.
The 100 pieces of data – 10 each from 10 bouts – ran like clockwork with not a single statistical outlier! Bout by bout, the demands on the contenders gave no quarter. Surely there would be one slip! Yet at the end, Precision and Accuracy were a STATISTICAL DRAW – just as with the 20µL bout at the Whitehead Institute 2 days earlier. And when this information started to get out, the activity in the taverns and the think tanks, in the dusky machine shops where the coffee is not cappuccino and in the shiny glass-and-light festooned edifices of exceptionalism, de Tocqueville’s revered roots resiliently reigned.
A highly expert pipetting person used top-of-the-line conventional pipettors and tips with which he was very familiar, wielded with the epitome of good technique in aspiration and touch-and-drag dispensing that bring out the very best performance in those superb conventional pipettors. Yet with brand new Differential Pipettors and a different dispensing technique this Lincolnesque scientist generated data that was not only fully comparable in precision and accuracy – but was delivered contact-free, untainted by touch or drag or immersion! No turning back, upcoming competitions must peer deeper and even more clearly into the meaning of true accuracy and the unaddressed issues of speed and ease – all grounded realistically in unchanging human nature.
From 10µL down through 0.75µL, the Differential Pipettor fully matches the precision and accuracy of the Gilson Pipetman and Rainin’s finest – while dispensing contact-free!
20uL & 1uL Precision /Accuracy Bout with Gilson Pipetman Theatrical Version
Whitehead Institute Ploegh Lab Differential Pipettor 20µL re-match and 1µL challenge. At 20µL they are again toe-to-toe. At 1 uL, Challenger appeared down, but Manager’s overtime shadowboxing round enabled Challenger to confirm 1µL skills, elucidate the unintended ring handicap, establish undefeated status and graciously improve pipetting understanding and ring practices. Ringside reports were that at 1 µL, with the intentional absence of an active referee and passive Differential Pipetting manager, the challenger’s 3.5% CV appeared inferior to the French champion’s 1.5%. But when DP Manager Dr. Donald Schwartz added an overtime shadowboxing round in order to aspirate straight to 1-2 mm depth the challenger’s 1uL CV was confirmed at 1.5%. The now closed-circuit crowd understood and erupted. The heartwarming comeback stimulated and inspired immediate follow-up groundbreaking studies that elucidated common errors that most good pipetting people are probably making every day, along with enhanced clarity on the simple steps that give correct and optimal small volume pipetting results.
200 – 900 nanoliter study shows that correct aspiration and dispensing methodology improves accuracy and reflects an accuracy advantage of contact-free Differential Pipetting dispensing. A study performed May 20, 2015, in the Artel Calibration Laboratory in Westbrook Maine.
The Differential Pipettor aspirated throughout the 200 – 900 nanoliter range and dispensed both contact-free and by traditional touchoff-and-drag into the Artel PCS to give precision volume measurements. With any aspiration difference thus eliminated, the two different dispensing methodologies were isolated as the cause of differences in the pipetted volume that was transferred. This was done by dipping the tip during aspiration the correct shallow 1-2 mm and by dipping an overly deep 8 mm. The interesting results reflect the balance between tip internal liquid retention and tip external liquid wickoff that occur during traditional touchoff-and-drag dispensing, a balancing act believed to be absent with the cleaner and more accurate contact-free dispensing methodology of the Differential Pipettor. The very simple contact-free dispensing of the Differential Pipettor appears to give truer and more accurate results and to also be free from the user technique variation inherent to traditional touchoff-and-drag dispensing.
VIGOROUS MIXING Can Be Done with the Non-Differential Mode!
CLICK ON THE VIDEO TO SEE AN EXAMPLE
This means, for example, that you could use the Differential mode to precisely aspirate a tiny volume and deliver it (contact-free if you wish) to a much larger volume… and then use the non-differential mode to get super vigorous mixing!
This graphic shows the remarkable Differential Pipetting mechanism in a way that makes it easier to understand how it completely avoids the resolution-vs-flow dilemma of all conventional pipetting.
In SCIENCE 1-12, elegant low volume studies clarify basic pipetting principles. Unsurpassed precision and accuracy are shown from 20µL down through 0.75µL, with unique contact-free dispensing. The absence of outliers and short sampling stands out and frees operators from mental stress and duplicate and triplicate practices. Ease and speed for regular laboratorians is noted.
Be assured that the one on the right wrote the attached article. A laboratory scientist’s intense mind, eye, heart and hand coordination still does some things better than any automated robot – even as humanity fights for its life against the very artificial intelligence it created to serve it, while technocracy replaces natural laws with supportive marinates.To automate or not to automate, that is the question! The bad always comes in to “help” just like the good does. It falls to us to try to tell the difference while we still can.
Pipetting is the commonest laboratory test in the world, and to those millions of hand-and-strained-eye cyclers a day, Travis explains the fundamentals needed to understand how the Differential Pipettor empowers laboratorians – not by embedding more chips and kindred regulations but by shining the light on differential displacement and setting it free in your hand.
The article describes pipetting and “displacement” technology and how the Differential Pipettor eliminates the resolution/flow tradeoff that bedevils conventional pipetting and does clean, contact-free dispensing delivery. The article links to studies that Travis did with the Artel PCS in the Artel Quality Control/Calibration laboratory in Westbrook, Maine that cast new light on small volume pipetting principles and phenomena and the variability caused by technique-laden touchoff-and-drag delivery. The article shows that the Differential Pipettor gives precision and accuracy that cannot be beaten by the best of the industry standard pipettors in the most expert of all hands, but goes beyond that in contact-free delivery and ease.
This article takes the mystery out and shows the soul as well as the mechanics of the thing, an excellent piece of writing that is as charming as it is lucid and powerful. It could be your paw from which Androcles can withdraw the thorn if you but click a picture or the Science link to the actual article.
No Outliers Reduces Mental Stress and Saves Time and $$$
Click here or on the picture or on SCIENCE 15 to read the full fascinating report.
With the Differential Pipettor, you will NOT get outliers, you will not aspirate short samples, and you will not have failed aspirations. You will NOT need to check the tip each time for this anymore! The unique Differential Pipettor design eliminates any skinny piston or small seal. And that eliminates all the sealing problems and aspiration instability caused by the skinny pistons and tiny seals that conventional pipettors are stuck with for smaller volumes.
This will reduce mental stress and wasteful testing, including eliminating the need for routine duplicate and triplicate practices.
