Benchmarking 384 Well Plates for High Throughput PCR

Introduction
In order to assess the benefits of Corning Life Sciences's 384 well PCR plates for high throughput applications, we performed individual experiments to compare Corning polyolefin plates with comparable PCR plates from two other manufacturers (A + B). The plates were tested for cross contamination between wells, fluid loss due to evaporation and wicking, and consistency and quality of amplification signal across the surface of the plate. A range of reaction volumes from 5 to 25 µL was used. This Technical Note will provide the customer with a benchmark for determining reaction conditions and choosing between consumables for high throughput PCR applications.

Methods and Materials
This study compared 384 well PCR plates (catalog no. 6502), Aluminum Sealing Tapes-384 (catalog no. 6569) and Reusable Sealing Mats (catalog no. 3699) with comparable 384 well PCR plates from two manufacturers (A + B).

The Perkin-Elmer GeneAmp PCR Reagents Kit (part no. N801-0055) was used for all PCR reactions. The PTC-200 DNA Engine thermal cycler used with the 384 well interchangeable head (MJ Research) was used for PCR amplification. PCR product was quantified with the PicoGreen dsDNA Quantitation Kit from Molecular Probes (catalog no. P-7589) in Black, Polystyrene, 384 well Solid Assay Plates from Corning Incorporated (catalog no. 3710) using an EG&G Wallac Victor Multilabel Counter (catalog no. 1420).

Reaction Volume Loss Due to Evaporation
Loss of PCR reaction volume due to evaporation and other causes must be a consideration in choosing plates and sealing materials. This assay was performed using Corning 384 well PCR plates and 384 well PCR plates from competitors A and B. Either Reusable Sealing Mats or Aluminum Sealing Tapes obtained from Corning were used with all plates as indicated. PCR reactions were prepared in accordance with the instructions contained in the Perkin-Elmer GeneAmp PCR Kit with the exception that the holding and melting temperatures were reduced from 94°C to 92°C. Two samples of each plate were used for each type of sealing material, and weighed before and after amplification.

Cross contamination between PCR reactions in adjacent wells of 384 well plates can be disastrous. To assay an occurrence of cross contamination on 384 well plates, alternating horizontal rows of each sample plate were filled with a 20 µL reaction volume of either blue or yellow dye. Any cross contamination between two wells would result in an easily discernible green well. Either sealing mats or tapes were used. Prepared and sealed plates were then subject to thermal cycling and visually inspected for cross contamination.

Quality of PCR Amplification Signal in 384 Well Plates Using Various Reaction Volumes
To assay the quality of signal under different reaction volumes, 384 well plates were prepared with 5 µL, 10 µL, 15 µL, 20 µL and 25 µL reaction mixes per well. Corning and competitor plates were used and both sealing mats and aluminum tape were applied. PCR reactions were prepared as above using the control template and primers provided in the Perkin-Elmer GeneAmp PCR Kit for amplification of a 500 bp region of bacteriophage Lambda. Six 5 µL samples were taken at random from each of the plates representing each reaction volume and examined on a 1% agarose gel. The gels were visually inspected for strength of signal, remaining primer and primer dimers.

Well-to-Well Consistency of Amplification Signal in 384 Well Plates Using Various Reaction Volumes
The remaining product in the plates after gel analysis was used in the PicoGreen dsDNA assay to determine well-to-well consistency of amplification of PCR product using various plates, sealing materials and reaction volumes. 5 µL of PCR product from the polyolefin plates were transferred to Corning Black 384 well Assay Plates containing 20 µL of 1X TE buffer. 25 µL of PicoGreen Reagent was added and the plates were incubated in the dark for three minutes. The fluorescence enhancement was read at 520 nm using the Wallac Victor at excitation 480 nm. Mean, standard deviation and coefficient of variation (CV) values were determined for every plate and sealing material combination at each reaction volume.

Results and Discussion
As discussed above, the purpose of this investigation was to assess the benefits of Corning 384 well plates for high throughput PCR using different sealing options and different reaction volumes.

The results were compared with 384 well PCR plates from two other manufacturers (A + B). Corning plates exhibited less than 1% evaporation of reaction volume as compared to as much as 3% with two other brands of plates. It should be noted that Corning brand Aluminum Sealing Tapes-384 and Reusable Sealing Mats were used with competitor brand plates. Other sealing materials may give different results. Likewise, Corning 384 well plates showed no cross contamination while the competitors' products showed as many as 7% of wells in a single plate displayed contamination from adjacent wells.

High throughput in molecular biology exponentially increases the number of assays and reactions a scientist can perform daily. As a result, reaction volumes must be smaller to contain costs. We tested various PCR reaction volumes, from 5 to 25 µL, to determine the smallest volume possible with an acceptable quality of signal. Smaller reaction volumes (5 – 15 µL) actually gave a stronger, cleaner signal than 20 – 25 µL volumes in 384 well plates as determined by visual inspection of 1% agarose gels (results not shown). These wells can be filled with fluid to a maximum volume of 25 µL; presumably, vapor pressure plays a role in determining adequacy of seal and therefore reaction temperature.

When fluorescence data from all 384 wells on a plate/sealing material combination was analyzed, it was found that Corning plates using either reusable mats or aluminum sealing tape gave coefficient of variations of under 20%. It should be pointed out that liquid handling was done by hand and involved two transfers: from master mix to PCR plate and from PCR plate to assay plate. All benchmarking of 384 well PCR plates must be examined in light of the consistency of liquid handling apparatus. With those considerations in mind, the well-to-well consistency is more than acceptable. Competitors A and B plates had results with CVs approaching 50%.

Currently, we are investigating Corning 384 well plates with various liquid handling robotics as well as other applications (i.e., high throughput sequencing). Benchmarking plates under these conditions will be reported in a future Technical Note.