New sealers for microplates and their areas of application in molecular biology and cell culture

Contents
1. EASYseal^™
The polyester sealer for everyday laboratory needs

2. SILVERseal^™
Heat resistant, pierceable aluminium sealer for PCR and sample storage

3. VIEWseal^™
Heat resistant, highly transparent sealer for precise optical measurements (fluorescence, realtime PCR)

4. POWERseal^™
Heat resistant, pierceable sealer for PCR

5. BREATHseal^™
Breathable sealer for cultivating bacteria, yeast and cells in microplates

6. Ordering information

Tightly sealed microplates are a basic prerequisite for their use in molecular biology, high-throughput screening, or cell culture. Three different sealing methods are commonly used at present:

1. The heat-sealing of microplates;
2. The sealing of microplates with CapMats;
3. The sealing of microplates with adhesive sealers;

Heat-sealing is the most commonly used method, especially when sealing polypropylene storage plates for storing active substances in high-throughput screening. Heat-sealing has the disadvantage that the microplates are ultimately destroyed by the sealing process and the samples are heated in the wells. Heat-sealing can be critical for cells or temperature-sensitive samples. In addition, heat-sealing requires the acquisition of an appropriate device, which is comparatively expensive for small research groups.

CapMats are also extremely popular for sealing microplates, especially in molecular biological applications such as PCR. The reuse of CapMats is widespread, but entails the risk of cross-contamination from carry-over of sample material. In order to avoid the disadvantages of heat-sealing and of CapMats, the use of adhesive sealers is an interesting alternative and the risk of cross-contamination is minimised by their application as disposable articles. Five different sealers, which can be divided into two different classes on the basis of the adhesive used, are supplied by Greiner Bio-One (Fig. 1).

Classical sealers such as EASYseal^™, SILVERseal^™ and BREATHseal^™ are coated with an acrylate adhesive. "Advanced sealers“ such as VIEWseal^™ and POWERseal^™ are coated with a pressure sensitive silicone adhesive. The silicone adhesive only adheres when pressure is applied to the foil surface.

The sealers are thus easy to work with, even in applications in which gloves have to be worn (the foil does not stick to gloves). Neither do substances in powder form or widely used model organisms such as Drosophila melanogaster and Caenorhabditis elegans adhere to vessels sealed with VIEWseal^™ or POWERseal^™.

Technical characteristics of thesealers

1. Adhesive force / evaporation
The better a sealer adheres to a microplate, the more tightly the microplate is sealed. The adhesion of the five sealers was determined using the example of 96 well microplates (Fig. 2). The adhesion of the sealers is characterised by the force in Newtons per mm (N/mm) that has to be applied in order to remove a sealer from a microplate.

The two sealers VIEWseal^™ and POWERseal^™ coated with silicone adhesive show the greatest adhesive force, followed by the classical aluminium sealer SILVERseal^™. Accompanying the adhesive force, microplates sealed with VIEWseal^™, POWERseal^™ or SILVERseal^™ have a low rate of evaporation (Fig. 3).

2. Optical properties of VIEWseal^™, POWERseal^™ and EASYseal^™
VIEWseal^™ differs from POWERseal^™ and EASYseal^™ in its exceptionally high optical transparency (Fig. 4), even in the shorter wavelength range (< 400 nm). The exceptional transparency of VIEWseal^™ is accompanied by a low level of autofluorescence and its property of minimal light polarisation.

VIEWseal^™ is therefore ideally suited for applications with sensitive optical detection systems, such as realtime PCR or fluorescence measurements in highthroughput screening. The signals to be measured are not falsified by high autofluorescence or light absorption of the sealers (Fig. 5, Fig. 6). A further area of application for VIEWseal^™ is the coverage of microplates for protein crystallisation (Fig. 7).

The individual compartments of the microplate in which crystallisation takes place are reliably and tightly sealed. As a result of the optical properties of VIEWseal^™, the development of crystals can be reliably detected and the crystals can be harvested properly since the sealers can be removed from the plate without jerking.

POWERseal^™ is characterised by a structured, pierceable surface and recommended for classical PCR. EASYseal^™ is an alternative to polystyrene lids and can be used for protection against evaporation and contamination.

3. Use of different sealers in PCR
The sealers VIEWseal^™, POWERseal^™ and SILVERseal^™ can be used in PCR. They are heatresistant up to 110°C and cold-tolerant down to -80°C. In a comparison of the yield, the fragment sizes, and the number of false bands between PCR products from individual vessels and PCR products from 96 well polypropylene PCR microplates sealed with sealers, no differences were found.

4. Use of BREATHseal™ for cultivating bacteria, yeast and cells
In many research projects in molecular biology, bacteria, yeasts or cells are cultivated in individual vessels such as test tubes, Erlenmeyer flasks or 50 ml centrifuge tubes. The aim is usually to isolate genomic DNA or plasmid DNA for sequencing or to isolate an expressed protein for crystallography.

With increasing automation and an increasing number of samples to be processed, 96 well MASTERBLOCK^®s and sometimes also 384 well microplates are used for cultivating the organisms.

For a high cell yield, in other words a high DNA or protein concentration, an optimal supply of the organisms with nutrients and oxygen is necessary. Especially the supply of oxygen is limited in microplates closed with sealers. The use of a gas permeable sealer such as BREATHseal^™ improves cell growth significantly (Fig. 9).

BREATHseal^™ is a gas permeable membrane coated with an acrylate adhesive, which consists of heat-welded rayon fibres. The pore size ranges from 10 to 50 µm (Fig. 10, next page). As a result of the layering of the pores, the membrane acts like a filter and provides reliable protection of the culture medium against aerobic bacteria, while ensuring optimal oxygen supply. A strongly adhesive sealer additionally facilitates the use of a microplate shaker.

Although hardly any liquid flows are visible in the wells, the cell yield is highest in microplates incubated on a shaker, regardless of the sealer used (Fig. 11).

5. Chemical stability / Temperature stability
The chemical stability of sealers is a major quality criterion for many areas of application of the sealers. For testing the chemical stability of the sealers, polypropylene microplates were filled with twelve different solvents (Table 1) and sealed with different sealers. The sealed microplates were turned upside-down so that the sealers or the adhesive were in direct contact with the solvents. The stability of the sealers was determined by visual assessment after one week (Table 1). As a result of the selected test method, BREATHseal^™ could not be included in the test, since the escape or evaporation of the solvent (gas permeable membrane) prevented any clear assessment. The highest stability was shown by POWERseal^™. Eight of twelve solvents tested had no influence on the sealer.

The temperature stability of the sealers is summarised in Table 2. SILVERseal^™, VIEWseal^™ and POWERseal^™ can be used in both PCR (temperature resistant up to 110°C) and for sample storage (temperature resistant down to -80°C). In the case of BREATHseal^™, especially at higher temperatures, the increased evaporation rate must be taken into account (gas permeable membrane structure of the sealer).

Table 1: Chemical stability of the sealers

Table 2: Temperature stability

Ordering Information

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