Normalization of 96 Samples Manually versus with WellAware^™ Electronic Protocols

Abstract: It is often necessary in molecular biology, forensic, research or other life science laboratories to normalize PCR, DNA, RNA, and protein samples to a uniform concentration. Normalization of these samples involves pipetting variable amounts of the initial sample and a buffer or water into microplates or vials. BioTX’s WellAware™ Personal Pipetting Systems, designed to eliminate errors, enable a researcher or analyst to pipet into 24, 48, 96, or even 384-well microplates accurately by using voice and light-guided software to illuminate wells placed directly on the WellAware screen. The illuminated wells can cut the time of all pipetting tasks by up to 50% and more. In this study, the speed of pipetting and some of the flexibility parameters for the WellAware^™ Personal Pipetting system (http://www.biotx.net) are demonstrated.

Objective: To simulate the pipetting steps necessary to bring 96 samples to a uniform concentration, time these steps, repeat the same steps using WellAware^™, and compare the speed of each procedure.

Procedure: A spreadsheet was created with 96 random concentration values that could be normalized to a value such that no more than 100 uL of total volume would be pipetted.

Example:

PART 1: MANUAL PIPETTING (without WellAware^™)
A 50 mL test tube containing water was used as the source to pipet each designated water volume into the appropriate wells of an empty 96-well destination plate. A stopwatch was started when pipetting began. No tips were changed; however, each volume was adjusted on one of the two pipettors such that volumes less than or equal to 20 uL were pipetted with the P20 and volumes greater than 20 uL were pipetted with the P200 pipettor. The water was pipetted in the order of the plate by columns (A1, B1 …H1, A2, B2…etc.).

Next, the water in the filled 96-well plate was used as the source to pipet each designated “sample” volume into the appropriate wells of the destination plate used above. Tips were changed each time to simulate the actual activity necessary when normalizing different samples. The stopwatch was stopped after all steps were completed.

PART 2: WELLAWARE PIPETTING
A. The WellAware^™ Cherry Picking GUI was used to create the normalization protocol. Every water and sample volume was entered into the electronic SOP (eSOP) exactly as in the previous spreadsheet. (Note: 0.001 was entered for 0.) The 96-well destination plate used above was emptied, and the 96-well “sample” plate was refilled with water. The 50 mL test tube was used as a non-barcoded source for the water pipetting into the empty 96-well plate which was placed on the right (Destination) side of the WellAware screen. Then, the water-filled “sample” 96-well plate was placed on the left (Source) side of the WellAware screen for sample pipetting. WellAware highlighted each Source and Destination well according to the eSOP and displayed the volume and well location on the pipetting screen.

B. The WellAware eSOP pipetting steps were sorted such that the water volumes were pipetted in increasing order (values 0.4 uL to 100 uL) and the sample volumes were sorted in increasing order, enabling the operator to save time dialing volumes into the pipettors. This is accomplished in WellAware by using the Sort button on the eSOP screen to first sort the spreadsheet by the volume column and then by the Sample name column. (Or open a second Excel worksheet, copy the values there and sort as you would normally sort in Excel using the Data-Sort commands. Then, copy the spreadsheet back into WellAware.) WellAware illuminated the correct diluent and sample wells corresponding to the eSOP well positions and sorted volumes. The same normalization volumes were used as before.

Results:

A. Manual Pipetting vs. Pipetting with WellAware

Pipetting with WellAware was 13 minutes and 50 seconds faster to complete the entire 96-sample normalization. The water steps alone were 6 minutes 18 seconds faster using WellAware. This is a time savings of almost 23%. Accuracy is not addressed in this experiment, but other studies show a large error reduction.

B. Pipetting with WellAware Sorting the Volumes

Pipetting with WellAware after sorting the volumes in increasing order was 36 minutes and 3 seconds faster than normalizing the samples without using WellAware. It was 22 minutes and 13 seconds faster than using WellAware without sorting to complete the entire 96-sample normalization. The water steps alone were 9 minutes 5 seconds faster using WellAware with sorted values and 15 minutes 23 seconds faster than without WellAware. That is a 59.5% time savings in the overall normalization process using WellAware versus without it.

PART 3: FUTURE PERSONAL AUTOMATION
Integration with an electronic pipettor/dilutor/dispenser with sample and dilution ranges of 50 nanoliters to 1000 microliters will permit full normalization of each sample with one pipet tip. The microliter sample would be placed on a NanoDrop, Picodrop, LabelGuard, NanoPhotometer or NanoVue spectrophotometer and the O.D. reading sent to WellAware. Normalization volumes would be automatically calculated by WellAware, which would immediately reset the pipettor to pick up the correct volume of sample. Moving to the lighted destination plate, the dilutor would mix and dispense the calculated volume of diluent and the sample.

For 96 well normalizations, the table of all sample readings would be transferred to WellAware, and then individual samples would be normalized as above.

Discussion: Normalization of samples using error-free WellAware^™ is more than twice as fast as pipetting without using the light and voice-guided software and specialized screen. WellAware^™ works with standard microplate formats but is easily customized for a variety of laboratory pipetting needs, including those associated with PCR, Genotyping, Sequencing, Sample Sorting, Cherry Picking, or Normalization. It has been proven to improve pipetting times as well as accuracy, and WellAware integrates with LIMS and Lab Instrumentation such as real-time PCR (RT PCR), NanoDrop^® and other spectrophotometers, and microplate readers. Electronic Standard Operating Procedures, or eSOPs, may be saved for review, retrieval, or repetition. The entire cost of a WellAware system is a fraction of that for an automated liquid-handling robot. WellAware may be placed inside biohazard, PCR, or tissue culture hoods too. Further studies with WellAware will contribute to error elimination, reduction in costs, and improvement in laboratory profitability or productivity.

BioTX Automation, Inc. contact information:

BioTX Automation, Inc.
16753 Donwick Dr., Suite A-6
Conroe, TX 77385

877-275-2468
877-ASK-BioTX
www.biotx.net
info@biotx.net

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