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E XTREME U NIFORMITY in P LASTIC D ISPOSABLES: BIOplastics' Story  

 

Extreme Uniformity (EU)
The Gold Standard for PCR Application

The BIOplastics' EU (Extreme Uniformity) products offer many unique features that distinguish them from all other comparable products in this market segment.They are truly the Gold Standard for PCR disposables. 
                   
The Design
We will never settle for a me-too product. Because we are molecular biologists ourselves, we know what the applications and the drawbacks of PCR disposables are. We want to provide an innovative and high-quality product that solves problems and helps scientists.

The Raw Material
Our design team has chosen the highest quality virgin polypropylene, thus preventing any metals, or other contaminants that influence biological experiments, to be part of the end product. Non-virgin or recycled polypropylene should only be used for less sensitive products like racks and boxes. Our design team also specified a special blend of virgin polypropylenes, instead of using a standard mix, and injection moulding to extreme tolerances. This results in PCR tubes, strips and plates that exhibit the perfect balance between transparency, softness, robustness, anti-statics and gas tightness.

 

The Injection Moulding Process
To injection mould a perfect product, a perfectly balanced mould is essential. Balancing a mould is a pure art in itself. It involves finding the trade-off between the injection temperature of the polypropylene, the temperature of the mould during the process of polypropylene solidification, and the production and positioning of the compartments that form the final product. BIOplastics has the in-house expertise to balance moulds precisely and effectively, producing an excellent disposable.

The Uniformity
Uniform wall thickness is a necessity to ensure accurate temperature transfer across the tube-wall barrier, thus providing an even and precise cycling reaction in the tube. BIOplastics guarantees a maximum wall thickness difference of 0.05 mm within a single tube, as well as between different tubes, tube-strips or plates.

The Angle
Measurements of cycler blocks of many brands in the market place show substantial differences in the angles of the holes. This means that a tube, tube-strip or plate, when placed in a block, will always have a different fit. BIOplastics has slightly modified the regular angle of the tubes to improve the surface contact in the blocks of all main brands of cyclers (new or used). It goes without saying that a good block-tube contact is of vital importance for a good thermal cycling reaction.



Evaporation and the Cap of the EU Tube
The amount of evaporation is not just determined by the quality of the polypropylene thin-wall barrier, but is also influenced by the way the cap closes onto the tube or plate. In contrast to closures of regular tubes, our closures have an extra sealing band as an additional evaporation barrier. However, due to the extreme tolerances to which our tubes, plates and caps are manufactured, our EU caps actually close much more easily. In addition, our EU tubes, plates and caps have dramatically lower evaporation rates that allow the use of reaction volumes as small as 5
µ l. In general, a dome cap offers better closure in comparison to a flat cap, because of the way the pressure is transferred. Finally, an anti-contamination tab prevents touching the inner walls of the tube during the process of opening. A small separate frosted writing area can be used to mark and identify the tube.

Optical Features
Both the single EU tubes, as well as the strip-caps for the EU strip and the EU plate are ready to be used for optical experiments like Real-Time PCR. The single EU tube has an optical, flat area on top of its dome shape; the strip-caps exhibit glass-grade, extra large thin-wall areas. All EU strips and plates are available in non-transparent white or black versions.


 The Evaporation Story, a Subtle Balance

 

Permeability of the Tube Wall
If you choose a certain type of thin-wall tube, strip or plate, you will always have to take into account the amount of fluid you are willing to sacrifice during your reaction because thin polypropylene is actually porous to water vapor. The permeability (= the amount of volume lost through the wall of a tube, strip or plate) is in close relationship with the wall thickness and the uniformity of the wall of that tube, strip or plate. Especially in PCR reactions without oil (so with heated lid) this loss can be substantial when using thin-wall tubes, strips or plates. However, using thin-wall tubes, strips or plates results in a better heat transfer and in a higher speed of reaction, so most of the time they are preferred over regular (thick-wall) tubes (that exhibit no evaporation through the wall).

The Effect of the Tube Cap
Other important factors to consider are the quality and specifications of the caps of tubes, and/or the closure of strips and plates. The way a cap closes onto a tube largely determines the leakage of gasses, aerosols and fluids through the top of the tube, during a cycling reaction. It is therefore highly essential to close tubes carefully, but without too much pressure because this will also cause leakages. The role of the heated lid on a PCR machine should not be underestimated: one should apply just enough pressure and certainly not use temperatures above 121oC
, because this will change cap/tube material characteristics. For PCR thermal cyclers that do not have an adjustable heated lid, special adaptors are available.

 

Quality aspects
A tube should be produced of the highest quality virgin polypropylene available. It should be soft and flexible thus providing a good closure. Take care with autoclaving EU tubes because if it is done too harshly the material will become brittle and not form a tight seal (max. 15 min at max. 121o
C and max. 2.2 bar). Sterilizing tubes by means of 60Co radiation can cause the material to age and loose its flexibility.

Evaporation grades
BIOplastics' PCR tubes, strips and plates are extensively tested for their evaporation properties. Evaporation is in general a function of the PCR volume; the lower the volume, the higher the evaporation will be. BIOplastics' PCR tubes, strips and plates are categorized into 3 different groups of so-called evaporation grades: 1, 2 and 3. These evaporation grades are based on actual measurements. Our EU products have passed grade 1 certification for evaporation.

Evaporation Grade 1:
Loss of PCR volume is less than 3%. It is possible to PCR in as less as 5
µl.

Evaporation Grade 2:
Loss of PCR volume is in between 3 and 15%. It is possible to PCR in as less as 15 µl.

Evaporation Grade 3:
Loss of PCR volume is more than 15%. It is recommended to use at least 20 µl
for your PCR.


 Quality Aspects of Plastics

What is sterility?
A sterile product is a product that is free of any living micro-organisms. Sterilization of BIOplastics is, in general, done by means of 60Co irradiation. However, irradiated products age faster than non-irradiated products. They are less flexible and break easier, because 60Co irradiation slightly influences the characteristics of the polymer material. Autoclaving the product yourself is much
softer for the plastic, but when done several times it will also harden the polymer. One could argue whether it is necessary to autoclave or irradiate PCR disposables at all; BIOplastics are produced under clean room and “no-hands-on” conditions.

What is DNa(se), RNa(se) and Pyrogen-free?
Producing products under strict clean-room and “
no-hands-on ” conditions ensures the absence of DNA, RNA, and their breakdown enzymes. To guarantee they are free of these contaminants, we test our DNA(se), RNA(se) and Pyrogen-free products. Customers should understand that a sterile product is not necessarily DNA(se), RNA(se) and Pyrogen-free.

What is metal-free?
The absence of metals in polymer products like tips and tubes is essential for laboratory use. BIOplastics' tips and tubes are made of virgin polypropylene of the highest quality available. No mould releasing agents are used during this process. This is possible because the moulds (the template shape in which the molten polypropylene is injected) are manufactured precisely, enabling an easy release when the product is solidified.

Low-cavity moulds versus Multi-cavity moulds
If you want a certain tip, tube or plate to have the highest uniformity available, a low-cavity mould should be used in its production. This implies that a few tips, tubes or plates are made at a time, and after that the next few are produced. On top of that, during release from the mould, each injection of product is collected in one bag, resulting in batches with exactly the same specifications. This takes more time, and is more expensive than using multi-cavity moulds from which 32, 64 or even 128 tips or tubes are released in one step. However, these tips and tubes differ heavily in specification and quality because a multi-cavity mould is less precise than a low-cavity one. Also, controlling the process of injecting the polymer into a multi-cavity moulds (pressure/temperature) is much more difficult. It is nearly impossible to balance such a mould. This can result in batches of tips or tubes or plates with differences in wall-thickness or orifice, thus destroying the reproducibility of your experiment. BIOplastics' tubes and plates are only manufactured with Low-, Semi-Low cavity moulds.


 The Importance of Filter Material

Filtertips are generally accepted as the solution in the prevention of cross-contamination in PCR reactions. A main source of this contamination is the formation of aerosols in the shaft of the pipette used during the PCR liquid handling. Carry-over of previously pipetted DNA can cause false positive signals.

Filter materials
The use of a filter in a filtertip eliminates the formation of aerosols in the shaft of the pipette, because it blocks the air-to-liquid interface between the sample and the shaft. During the last 10 years this simple idea has evolved in numerous types and brands of filtertips and filter materials.

In earlier days filters were made of cellulose. Not being inert, this material was not suited to prevent cross-contamination in sensitive pipetting procedures. Nowadays most filters are made of 3-dimensional cross-linked polyethylene, which is totally inert.

 

Self Sealing
The most important parameter for a filter tip is the pore size of the applied filter material. The pore size must be smaller than 25 µm to protect against aerosols at all. If the pore size is between 20 and 25
µ m, a filter will give some protection, however liquid will pass when overloaded. Below 18 µ m, a filter will give superior protection. When pore sizes get below 14 µ m, it becomes too difficult to pipet liquids in a reproducible manner, because of the airflow barrier. Therefore, it makes no sense to use filters with this small pore size.

The costs and investments to manufacture a filter material with an average pore size of 16 µm are very high. Only the BIOplastics' Self Sealing Non Collapsing (SSNC) filter tip meets this specification. This filter material is patented under US Patent #5364595. It is the perfect balance between superior protection and accurate pipetting.

Filtering Capacity
The filtering capacity of a certain filter is defined as the ratio of filter length and pore size. The longer a filter is, the better it filters. This relation is almost linear. BIOplastics' filters have the longest length available in the market when compared to other brands. On the other hand, the smaller the pore size is (down to 14 µ
m), the better it protects. When these two factors are combined, the result is a superior filter tip.

 

Non-Collapsing or Collapsing?
In some applications (like radioactive experiments) it is unacceptable that a liquid can pass through a filter at all. That is why some filters (the so-called collapsing filters) are chemically treated with powder or other chemicals. When the liquid touches this filter, it will collapse. One is not able to dispense the sample anymore. It will also be contaminated and unusable. However, optimal protection is ensured, because neither aerosol nor liquid has passed the barrier. The BIOplastics' Self Sealing Collapsing (SSC) filter tip is therefore a very specific choice. It uses the same, patented Self Sealing filter material as described above.

Autoclaving SSC and SSNC filtertips
It is not possible to autoclave Collapsing (SSC) filtertips. However, the BIOplastics' SSNC filtertips can be autoclaved when desired (15 min at 121oC and 2.2 bar, then dry for
3 hrs at 80oC). It is important to be aware that during autoclaving the gaseous water phase will fill the pores of the filter, resulting in a water filled matrix after cooling down. With SSNC filters one has to be sure that the drying time is long enough to allow complete evaporation. Water filled pores will affect the speed, accuracy and reproducibility of the pipetting. It may be worth questioning the need of using sterile filtertips for PCR. Another option would then be the purchase of sterile SSNC filtertips instead of autoclaving them yourselves. 

Two BIOplastics filters?.
The choice is easy and explainable: Self Sealing Non Collapsing (SSNC) filtertips for optimal protection, or Self Sealing Collapsing (SSC) filtertips for complete security in special applications.

All PCR Plastics made in Netherlands

For more information about PCR products inquire at info@eENZYME.com .