GB2453432A

GB2453432A - Thermal cycling applications vessel

Info

Publication number: GB2453432A
Application number: GB0817862A
Authority: GB
Inventors: Simon May; Jeffrey Leonard Coulling
Original assignee: Advanced Biotechnologies Ltd
Current assignee: Advanced Biotechnologies Ltd
Priority date: 2007-10-02
Filing date: 2008-10-01
Publication date: 2009-04-08
Also published as: GB0817862D0; GB0719193D0; WO2009044196A1

Abstract

A vessel for thermal cycling applications, the vessel being formed from a thermoplastic material such as polypropylene and comprising at least one well 12 for containing a sample, the well 12 comprising an upper wall portion 14, an intermediate wall portion 15 a lower wall portion 17 and a bottom 16. The lower wall portion 17 corresponds to the region of the well intended to contain, in use, a sample, wherein the wall thickness of the lower wall portion 17 is less than the wall thickness of the intermediate wall portion 15 and wherein the wall thickness of the lower wall portion 17 is between about 0.06mm and about 0.17mm. The relatively thin wall section allows rapid heat transfer. There is also an independent claim to a method of manufacturing such a vessel by injection moulding.

Description

A VESSEL

Field of the Invention

The present invention relates to plastic vessels such as single tubes, tube-strips and multiwell plates used as containers for chemical or biological reactions, such as polymerase chain reactions (PCR) or for storage of chemical or biochemical samples, and to methods of manufacturing such vessels, it is particularly applicable, but in no way limited, to plastic vessels with ultra-thin wall portions allowing for efficient heat transfer in apparatus for thermal cycling applications, such as apparatus used in PCR reactions, and to methods for manufacturing such vessels.

Backround of the Invention Polymerase chain reaction (PCR) is a thermal cycling procedure which employs the use of plastic vessels such as single tubes, tube strips and multiwell plates.

The purpose of the PCR thermal cycling process is to amplify DNA by reproducing identical DNA strands in very small quantities to obtain larger samples of identical DNA for further experimentation for example. PCR uses a thermally controlled enzymatic reaction in order to form identical copies of double stranded DNA found in very small quantities in the original sample. The copies formed are then used to form further identical copies and so on in a chain reaction.

In most instances PCR needs the sample to be amplified and the other reaction components to be seaied in a reaction vessel wherein the sample is incubated at several different temperatures for the various reaction steps. These steps may include denaturation, hybridisation and extension. The denaturation step involves the separation of the initial double-stranded DNA into single stranded templates for use in the hybridization and extension steps. It is very important during these incubation steps that the sample is retained very accurately at a given temperature for a given period of time, and that these cycles can be accurately repeated a given number of times.

It is preferable that the temperature change between the two incubation steps can be effected rapidly. This is important since each of the steps utilizes enzymes which operate only within a given optimal temperature range and as such generates a much more efficient reaction at these optimal temperatures.

In addition, the overall time for an amplification procedure is determined by the length of time the reaction mixture needs to be held at each incubation temperature, plus the time required to change between the temperatures. It is important for processing times to be optimized so that the greatest number of procedures can be carried out. This can be done by reducing the time needed to change between incubation temperatures.

Typically, PCR temperature control takes place by one of two methods, The first of these methods is the use of a thermal cycling instrument which comprises a highly thermally conductive sample block. The sample block is formed such that it has a geometry which matches the reaction vessel containing the reaction mixture, typically a series of wells. Due to this matching geometry there is contact between the wall of the reaction vessel and the sample block which results in good neat transfer between the sample block and the reaction mixture.

Historically, the minimum cycle time limitation of PCR using these thermal cycling instruments has been determined by the thermal mass of the sample block and related heat pump components of the system. However, due to recent improvements in some of these thermal cycling instruments, which have decreased the cycle time of the sample block, the rate limiting step is now considered to be the thermal mass of conventionally moulded plastic PCR vessels, The second of these methods is less widely used and consists of an instrument with a series of fixed temperature liquid baths. The PCR vessels are automatically moved between these baths according to the PCR temperature requirements. This method is generally found in high throughput situations where a large number of samples are processed simultaneously. Since this method involves contact between the vessel and the liquid in the bath, the rate determining step of the PCR process is considered to be the thermal mass of the plastic PCR vessels.

to There are a number of PCR vessels available commercially adapted for use in different thermal cycling machines. Such vessels include single tubes, tubes arranged in strips and tubes arranged in arrays. The tubes arranged in arrays, known as multiwell plates, have become the most widely used vessel type for PCR and thermal cycling in general. Such multiwell plates can be formed by a number of methods which include: 1. Heating a sheet of plastics material until it is soft and then moulding it to a preformed shape. Such methods are cheap and produce vessels with a low thermal mass and very thin tube wall thicknesses. However, the method also results in plates with little structural integrity and due to this lack of rigidity cannot be used in automated processes. In addition there is little control over the way the plastic stretches which can result in non-uniform tubes, uniformity being very important in PCR processes, such that standards can be developed.

2. High pressure injection moulding is the second method available, which is extremely popular for a number of reasons, which include: a) consistent wall thickness which can typically be in the order of 0.23mm; b) increased rigidity of the plates using duel step injection moulding with one plastics material for the frame of the plate and a different plastics material for the wells or tubes.

A number of multiwell plates exist in the market which are marketed as having thin walls of around 0.23mm to 0.38mm. However, these walls are still not thin enough to take full advantage of the faster cycle times available with the latest thermal cycling instruments.

A number of attempts have been made to produce multiwell plates with thinner wall thicknesses of the wells/tubes. One such product is formed by separately vacuum forming the wells and injection moulding the frame and joining the two elements together to form the plate.

This particular offering, however, is only made to fit the manufacturer's own instruments, but does offer wells with wall thicknesses of around 0.09mm but with little structural integrity and as such the tubes can be easily crushed. Such products are also more costly to produce than standard injection moulded is plates, and since such products are generally only single use, this plays an important part. Thus wholly injection moulded plates are preferred. However, the most injection moulding friendly plastics material at the moment, is considered to be polypropylene. However, plastics formed from the polypropylene can only be formed with wells with wall thicknesses of 0.18mm to 0.23mm due to the limitations of the injection moulding process itself.

Injection moulding generally involves two mould halves which, when brought together, form a mould cavity. Molten plastics material is then injected into this mould cavity to fill the mould cavity and is then allowed to cool and set before the finished product is removed from the mould cavity.

The molten plastics material may be injected into the mould cavity at a number of injection points, typically 6 in the case of a 96 well plate, which means that the plastics material has several "fronts" which meet up with each other inside the mould cavity. It has been found that when thinner walls are produced those fronts do not meet up and marry correctly, resulting in weak knit lines and holes, leading to inferior products if thicknesses less than 0.18mm are desired, This is because as soon as the molten polymer enters the mould cavity it begins to cool, and the viscosity of the molten plastics material thus increases. When the preferred plastics material polypropylene is used, it cools very rapidly and a skin of around 0.038mm begins to form, which effectively reduces the flow path further in the thin wall area, which leads to the above minimum wall thicknesses.

EP 1 618 954 (WIT EDWIN DE) teaches a tube of steel construction which has a biologically inert interior coating, for instance polymer. Steel-containing tubes are not beneficial because of the costs and complexity related to the manufacturing process.

DE 4022792 (EIGEN MANFRED PROF DR and SIMM WOLFGANG) and GB 806482 (NOBLE DENN1S ALEXANDER JOHN) teach various methods of forming a vessel from a film of polymer by stretching the film. While the resulting product is of total polymer construction, using these techniques, the tube wall thickness has to be relatively constant over the whole product. This limits their use significantly, as the starting polymer film thickness defines the thickness range of the whole product. Also, no complex shapes can be manufactured.

US Patent No. 5,922,266 (GROVE DALE) discloses a method of injection moulding, wherein the mould cavity is squeezed while injecting molten polymer into the cavity. The method aims at producing optically high quality articles such as contact lenses and plastic layers of optical discs. The optical properties of the article to be moulded are improved because of the reduced internal stresses of the product and reduced number of optical distortions due to the equalization of pressurization by the shrinking cavity and gradual cooling of the polymer. A similar method is disclosed in US 4,707,321 (SEGAWA TAKASHI and TSUBOI KUNIO). Neither of the documents relate to manufacturing of vessels for biological assays or exceedingly thin object portions in general.

One special kind of plastic vessel and its manufacturing process by conventional injection moulding is disclosed in WO 2004/054715 (CLARKSON JOHN MICHAEL and ANDREWS DAVID).

W020071029101 (TURNER BRUCE R) discloses a method of forming a thin walled tube by forming an oversized mould cavity from two mould members which are movable relative to each other, to effectively change the volume of the mould cavity. Molten plastics material is then injected into the oversized mould cavity and whilst the plastics material is still molten the mould members are moved to reduce the volume of the mould cavity, compressing the molten plastics material to form the vessels with ultra thin walls. The problem with this method is that specialized machines are required to form the vessels and conventional equipment cannot be employed.

It is an object of the present invention to overcome, or at least mitigate, some or all of the problems described above.

Summary of the Invention

According to an aspect of the present invention there is provided a vessel for thermal cycling applications, the vessel being formed from a thermoplastic material and comprising at least one well for containing a sample, the well comprising an upper wall portion, an intermediate wall portion a tower wall portion and a bottom, the lower wall portion corresponding to the region of the well intended to contain, in use, a sample which is preferably a PCR sample, wherein the wall thickness of the lower wall portion is less than the wall thickness of the intermediate wall portion and wherein the wall thickness of the lower wall portion is between about 0.06mm and about 0.17mm.

Preferably the lower wall portion relates to that region of the well wall defined by the lowermost in use up to about 30% of the volume of the well preferably about 1% to about 30%, more preferably about 5% to about 25%, even more preferably about 5% to about 20%, yet more preferably about 10% to about 20%, still more preferably about 10% to about 15% of the volume of the well.

Preferably the lower wall portion relates to that region of the well wall defined by the lowermost in use up to about 60p1 of sample volume of a well in a 200iJl 96 well plate or an individual sample vessel, preferably about 5p1 to about 6Opl, more preferably about lOpi to about 50p1, still more preferably about lOpI to about 4Opl, yet more preferably about I Opt to about 3Opl, most preferably about 20p1.

Preferably the lower wall portion relates to that region of the well wall defined by the lowermost in use up to about 15p1 of sample volume of a well in a 5Opl 384 well plate, preferably about lpl to about 15p1, more preferably about 5p1 to about I5pl, most preferably about lOpl.

Preferably the lower wall portion relates to that region of the well wall defined by the lowermost in use up to about l5OpI of sample volume of a well in a SOOpI individual sample vessel, preferably about lpl to about l5Opl, more preferably about lOpi to about lOOpl, still more preferably about 20p1 to about 80il, yet more preferably about 4Opl to about 6Opl, most preferably about 50p1.

Preferably the vessel is formed from a single step injection moulding process Preferably the thermoplastic material is polypropylene.

According to a further aspect of the present invention there is provided a method of manufacturing a vessel comprising the steps of: a) forming a mould cavity with a first mould member and an opposing second mould member; b) providing at least one injection point and providing at least one outlet port; c) injecting into the at least one injection point a molten thermoplastic material of a volume larger than the volume of the mould cavity; d) allowing the excess molten thermoplastic material to exit the mould cavity from the at least one outlet port; e) allowing the molten thermoplastic material to set; I) removing the excess thermoplastic material that exited the mould cavity from the outlet port from the vessel formed in the mould cavity; wherein the first and second mould members form a vessel mould cavity such that the vessel formed will have at least one well for containing a sample, the well comprising an upper wall portion, an intermediate wall portion a lower wall portion and a bottom, wherein the wall thickness of the lower wall portion is less than the wall thickness of the intermediate wall portion and wherein the wall thickness of the lower wall portion is between about 0.06mm and about 0.17mm, Preferably the lower wall portion relates to that region of the well wall defined by the lowermost in use up to about 30% of the volume of the well preferably 1% to about 30%, more preferably about 5% to about 25%, even more preferably about 5% to about 20%, yet more preferably about 10% to about 20%, still more preferably about 10% to about 15% of the volume of the well.

Preferably the lower wall portion relates to that region of the well wall defined by the lowermost in use up to about 60p1 of sample volume of a well in a 200pl 96 well plate or an individual sample vessel, preferably about 5p1 to about 6Opl, more preferably about lOpI to about SOpl, still more preferably about lOpI to about 4Opl, yet more preferably about lOpI to about 3Opl, most preferably about 2Opl.

Preferably the lower wall portion relates to that region of the well wall defined by the lowermost in use up to about I 5p1 of sample volume of a well in a SOpI 384 well plate, preferably IpI to about lSpl, more preferably about Spl to about l5p1, most preferably about lOpI.

Preferably the lower wall portion relates to that region of the well wall defined by the lowermost in use up to about lSOpl of sample volume of a well in a 500pl individual sample vessel, preferably about IpI to about l50p1, more preferably about lOpI to about lOOpI, still more preferably about 20p1 to about 8Opl, yet more preferably about 40p1 to about 60p1, most preferably about 5Opl.

Preferably the thermoplastic material is polypropylene.

Brief Description of the Drawings

Figure 1 illustrates a cross-sectional view of a vessel according to the present invention.

Figure 2 illustrates a cross-sectional view of a vessel according to the present invention with dimensions, those dimensions relating to a well suitable for use in a 200pl 96 well PCR thermal cycler. In this figure a corresponding numbering system to that of Figure 1 is used.

Detailed Description of the Invention

Preferred embodiments of the present invention will now be more particularly described1 by way of example only. These represent the best ways currently known to the Applicant of putting the invention into practice but they are not the only ways in which this can be achieved.

Figure 1 illustrates a well of a vessel according to the present invention. Such a vessel can comprise a single well or sample tube (12) with or without an attached cap, a one-dimensional array having a plurality of wells (12) arranged in a line (microtitre strip) or in a two-dimensional grid (multi-well plate). The well (12) has an upper connecting portion (10) which forms the deck or connecting portion of the strip or plate in those embodiments. The wells (12) protrude downwardly, away from and below the connecting portion (10), such that their openings (11) are on the upper surface of the connecting portion (10).

Generally the wells (12) are provided with a chimney portion or rim (13) extending from the upper surface of the connecting portion (10) in the opposite direction to the well (12). The chimney portion (13) allows for more efficient sealing of the well by known techniques such as heat sealing.

The wells (12) generally comprise an upper wall portion (14), an intermediate wall portion (15) a lower wall portion (17) and a bottom (16). The upper wall portion (14) generally extends downwardly from the lower surface of the connecting portion (10) and typically either the internal wall surface of this portion and/or the external wall surface of this portion extend(s) substantially at right angles to the general plane of the mouth of the well or normal to the plane of the deck or connecting portion. A generally frustoconical shaped intermediate portion (15) extends downwardly from the upper wall portion (14) in which the wall thickness reduces towards the bottom of the portion, and thus towards the bottom of the well. Reduction in wall thickness may be gradual as shown in Figures 1 and 2, or it may be in a stepped fashion, over one or more steps. This has the effect that the wall thickness of the well (12) is continuously reduced to the thin lower wall portion (17) where the wall thickness remains substantially constant before increasing in thickness as is merges into the bottom (16) of the well.

The wall thickness of the upper wall portion (14) is generally between about 0.23mm � 25% and about 0.76mm � 25%. The very thin wall portion (17) of the lower wall portion has a wall thickness generally between about 0.06mm � 25% and about 0.17mm � 25%, a preferred wall thickness of the thin portion is about 0.1mm � 25%. The reason for the high error margin is due in part to the natural variation in the wall thickness due to for example tooling variations and in part due to movement of the pins of the mould during the injection process wherein the pin may move such that one part of the wall is thicker than another part of the wall.

Due to the manufacturing techniques used to injection mould the multi-well plates there is a small amount of variation in the wall thickness of the walls of the wells. Although the wall thickness will remain substantially constant there may be some small variation therefore the thickness of the very thin wall portion should be taken to be the mean value of the internal distance between the outer surface and the inner surface of the wall portion in question.

In one embodiment, the wells (12) are arranged in a 96 well multi..well plate. A well according to these dimensions is shown in Figure 2. Generally, in the case of a 96 wall mutli-well plate, and other sample tubes designed to be used in a 96 well PCR thermal cycier, the full volume of the well is not used, Typically, in the case of a 200pI 96 well plate, only about 2Oil of sample is placed in the bottom of the well and the main contact between the sample block of the thermal cycling apparatus and the well is via the side wails (15) and not the bottom portion (16) of the well. Therefore, only the side walls in the vicinity of the sample need to be of a very thin cross-section as this is the area in which the heat transfer to the sample takes place. This allows for the remainder of the walls and the bottom portion to be formed with a standard thickness to impart the necessary rigidity to the wells (12). Similar logic concerning the amount of sample generally contained in the tube applies in the case of 384 wall plates.

The present invention provides methods for forming wells (12) with a very thin wall portion (17). The method generally comprises manufacturing the well (12) either as an individual well, as a strip or as a plate by a single process of injection moulding. A mould cavity is formed from a first mould member and a second opposing mould member, the mould members being connectable to a standard injection moulding apparatus. At least one injection point is provided for in at least one of the mould members. When, for example, a plate is being formed, six injection points may be provided in one of the mould members to allow for increased flow of the molten thermoplastic material in the mould cavity before the molten thermoplastic begins to cool.

At lease one outlet port is provided for in at least one of the mould members where the bottom of the sample tube will be formed. If a single well is being formed the mould member will have a single outlet port. If, however, a plate is being formed with multiple wells each well bottom may preferably be provided with an outlet port in the mould member, i.e. 96 wells require, 96 outlet ports.

Standard injection moulding techniques are then used to inject the molten thermoplastic material into the mould cavity using the injection points in the mould member(s). A greater volume of molten thermoplastic material is injected into the mould than is required by the volume of the mould cavity. The outlet ports provided allow the air to exit from the mould cavity and for the excess molten thermoplastic material to exit the mould cavity.

The excess molten thermoplastic material is collected in a reservoir and the molten thermoplastic material is allowed to set and can be removed as necessary (see below). As the molten thermoplastic material is injected into the mould cavity it begins to cool. Thus, molten plastics material flows over material which was injected into the mould before it, but which has solidified or partially solidified, This presents a very real problem when trying to mould components with very thin walls e.g. walls less than 0.2mm in thickness, The injection pressures required to overcome the constriction inside the mould caused by set or partially set plastics material within the mould become unattainable with conventional equipment. By arranging for only the wall portion associated with the sample-containing volume to have a very thin wall, normal injection techniques can be used. Such pressures do not affect the respective positioning of the mould members more than would normally be the case, This allows for substantially constant wall thickness in the very thin wall portion and the remainder of the well. In addition problems can occur with the meeting and joining of different fronts of plastics material, since the very thin walled portion is very small the plastics material does not have far to travel to meet the thermoplastic front coming from the opposite direction at the bottom. This means that it does not solidify as quickly and as such the two plastic fronts will be able to meet and mesh with each other, Once the molten thermoplastic material has set the excess thermoplastic material or spru can be removed from the bottom of the sample tubes to give the finished product.

As discussed earlier different manufacturers have different offerings for their multi-weU plates as there is a certain aniount of design freedom afforded to the designers of PCR plastic plates. They may design their multi-well plates such that they are only compatible with their own machines, or they may work to industry standards. Even when working to industry standards there is some room for design freedom in certain aspects of the multi-well plates. One such area of design freedom is the actual volume of the individual wells of the multi-woN plates. The reason for varying the well volume from offering to offering and from manufacturer to manufacturer is to provide the customer with a variety of volumes to meet the particular needs of the customer for various end applications of the multi-well plates. The 96 well plates generally have an individual maximum well volume of about 200p1 However the plates are formed in a number of different sizes and can vary across a wide range from about 5OiJl, to about 340p1. The 384 well plates generally have an individual well volume of about 40p1, however this can vary from about 25.ji to about The well volume varies according to the proposed end use of the particular plate in each instance.

Individual sample vessels, which may be stand alone or connected in strips also exist. These individual sample vessels generally have a volume from about 5Opl to about 500pl. Again the volume varies according to the proposed end use of the sample vessel in each instance.

For example in a standard 200p1 96 well plate, the present invention will provide a very thin wall portion in the lower wall portion to contain a minimum working volume of sample of about 5p1 to a maximum working volume of sample of about 50jl, preferably about lOl.il to about 4Opl, more preferably about l5p1 to about 30pt and most preferably about 20p1.

Table I shows individual volumes of wells of 96 well plates available commercially at the present time along with preferred working volume of sample to be contained within the very thin wall portion of the lower wall portion of the present invention along with the % volumes thereof.

Thus the preferred sample containing portion defined by the thin walled portion in a standard 200pl 96 well plate is from about 3% to about 25% of the total volume of the individual well.

In a standard 50pJ 384 well plate, the present invention will provide a very thin wall portion in the lower wall portion to contain a minimum working volume of sample of about IpI to a maximum working volume of sample of about 20p1, and preferably about Spl to about 1 Spl, more preferably about I OpI.

Table II shows individual volumes of wells of 384 well plates available commercially at the present time along with preferred working volumes of sample to be contained within the very thin wall portion of the lower wall portion of the present invention along with the % volumes thereof.

Thus the preferred sample containing portion defined by the thin wailed portion in a standard 5Opl 384 well plate is from about 2% to about 36% of the total volume of the individual well, preferably about 9% to about 27% of the total volume of the individual well, more preferably about 18% of the total volume of the individual well.

Table Ill shows volumes of individual sample vessels with preferred working volumes of sample to be contained within the very thin wall portion of the lower wall portion of the present invention along with the % volumes thereof.

Thus the preferred sample containing portion defined by the thin walled portion in a standard 500p1 individual sample vessel is from about 2% to about 36% of the total volume of the individual well, preferably about 9% to about 27% of the total volume of the individual well, more preferably about 18% of the total volume of the individual well.

As one might expect, these %, p1 and well wall thickness ranges should not be considered as absolute values and would themselves be subject to measurement variations of � 15% to � 5%, typically � 10% Maximum Well ______ ______ ______ ______ ______ Preferedworkingofurneof samole_ContainJg_portion _______ VoIumep 5 10 _15 20 25 _30 35 -45 -50 60 70 --80 _90 100 110 10% 20% 30% _____--------- -100 5% 10% 15% 20% 25% 30% _____- ------ 3% 7% 10% 13% 17% 20% 23% 30% _____------- 3% 5% 8% 10% 13% 15% i 23°, 2 30% --- 250 2% 4% 6% 8% 10% 12% 14% 18% 20% 24% -28% 32 ______ 300 2% 3% 5% ___ 8% 10% 2% 15% i7 20% 2 27% 3 ____ - 330 -2% 3% 5% 6% 8% 9% 11% 14% 15% 18% _____ 24% 27% _____ - -340 4% 6% 7% 9% 10% 13% 15% 18% _____ 243 26% 29% 32% -350 3% 4% 6% 7% 9% 10% 13% 14% 1 20% 23 2 31%

TABLE I

I Mxirnum Well ______ Preferred_working volume of sample containing portion ______ Volume jl 1 2 3 4 5 8 10 15 20 4% 4% 12% 16% 20% 32% _____--- -3% 7% 10% 13% 17% 27% 33% _____-- 3% 6% 9% 11% 14% 23% 29% _____-- -3% 5% 8% 10% 13% 20% 25% 38% _____ 2% 4% 6% 8% 10% 16% 20% 30% -2% 4% 5% 7% 9% 15% 18% 27% 36%

TABLE II

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N-(I)

*-0 I C) (4 N 0)

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---= -ô I U) Cl) 0 N-U) U) C') -0.J In

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0 1') 10 U) :C) C) -0 0) Ii) C) N.rr.-..-r.-II)': I) 1; C') 10': N 000)10 N-U) C') U) N 0 0) 0) 0) 10 N. CD C) 0 1') JO CO N-ID CD IL) U)':': C) (N 0Ifl010Wlg)u)': ::r')c') C)) (I) C) 0 N-U)': C) C) C') C) C') (N N C) N

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O Cl) C') C') I (N N - ,-s--r E 0 0 0 0 0 0 0 C) 0 0 0 E C) Cl) 0 Ct) o c') st It) C) U) 0 E-2 NNC)C)C)C),j-U) 1 i H;

Claims

CLAIMS: 1. A vessel for thermal cycling applications, the vessel being formed from a thermoplastic material and comprising at least one well for containing a sample, the well comprising an upper wall portion, an intermediate wall portion a lower wall portion and a bottom, the tower wall portion corresponding to the region of the well intended to contain, in use, a sample, wherein the wall thickness of the lower wall portion is less than the wall thickness of the intermediate wall portion and wherein the wall thickness of the lower wall portion is between about 0.06mm and about 0.17mm.
2. A vessel as claimed in Claim I wherein the lower wall portion relates to that region of the well wall defined by the lowermost in use up to about 30% of the volume of the well.
3. A vessel as claimed in Claim I or Claim 2 wherein the lower wall portion relates to that region of the well wall defined by the lowermost in use about 1% to about 30% of the volume of the well.
4. A vessel as claimed in any of the preceding claims wherein the lower wall portion relates to that region of the well wall defined by the lowermost in use about 5% to about 25% of the volume of the well.
5. A vessel as claimed in any of the preceding claims wherein the lower wall portion relates to that region of the well wall defined by the lowermost in use about 5% to about 20% of the volume of the well.
6. A vessel as claimed in any of the preceding claims wherein the lower wall portion relates to that region of the well wall defined by the lowermost in use about 10% to about 20% of the volume of the well.
7. A vessel as claimed in any of the preceding claims wherein the tower wall portion relates to that region of the well wall defined by the lowermost in use about 10% to about 15% of the volume of the well.
8. A vessel as claimed in Claim I wherein the lower wall portion relates to that region of the well wall defined by the lowermost in use up to about 6Opl of sample volume of a well in a 200pl 96 well plate or an individual sample vessel.
9. A vessel as claimed in Claim 8 wherein the lower wall portion relates to that region of the well wall defined by the lowermost in use about 5pl to about 6Opl of sample volume of a well in a 200pl 96 well plate or an individual sample vessel.
10. A vessel as claimed in Claim 9 wherein the lower wall portion relates to that region of the well wall defined by the lowermost in use about lOpl to about 5Opl of sample volume of a well in a 200pl 96 well plate or an individual sample vessel.
11. A vessel as claimed in Claim 10 wherein the lower wall portion relates to that region of the well wall defined by the lowermost in use about IOpl to about 40p1 of sample volume of a well in a 200pl 96 well plate or an individual sample vessel.
12. A vessel as claimed in Claim 11 wherein the lower wall portion relates to that region of the well wall defined by the lowermost in use about IOpl to about 3Opl of sample volume of a well in a 200pl 96 well plate or an individual sample vessel.
13. A vessel as claimed in Claim I wherein the lower wall portion relates to that region of the well wall defined by the lowermost in use about 2Opl of sample volume of a well in a 200pl 96 well plate or an individual sample vessel.
14. A vessel as claimed in Claim I wherein the lower wall portion relates to that region of the well wall defined by the lowermost in use up to about 15pl of sample volume of a well in a 50p1 384 well plate.
15. A vessel as claimed in Claim 14 wherein the lower wall portion relates to that region of the well wall defined by the lowermost in use about IpI to about l5pl of sample volume of a well in a SOpl 384 well plate.
16. A vessel as claimed in Claim 15 wherein the lower wall portion relates to that region of the well wall defined by the lowermost in use about 5pl to about I Spl of sample volume of a well in a 5Opl 384 well plate.
17. A vessel as claimed in Claim 16 wherein the lower wall portion relates to that region of the well wall defined by the lowermost in use about lOpl of sample volume of a well in a SOp1 384 well plate.
18. A vessel as claimed in Claim I wherein the lower wall portion relates to that region of the well wall defined by the lowermost in use up to about I 50p1 of sample volume of a SOOpI individual sample vessel,
19. A vessel as claimed in Claim 18 wherein the lower wall portion relates to that region of the well wall defined by the lowermost in use about lpl to about I 50p1 of sample volume of a 500pl individual sample vessel,
20. A vessel as claimed in Claim 19 wherein the lower wall portion relates to that region of the well wall defined by the lowermost in use about I OpI to about lOOpI of sample volume of a SOOpI individual sample vessel.
21. A vessel as claimed in Claim 20 wherein the lower wall portion relates to that region of the well wall defined by the lowermost in use about 2Opl to about 8Opl of sample volume of a 500p1 individual sample vessel.
22. A vessel as claimed in Claim 21 wherein the lower wall portion relates to that region of the well wall defined by the lowermost in use about 4Opl to about 6Opl of sample volume of a 500pl individual sample vessel.
23. A vessel as claimed in Claim 22 wherein the lower wall portion relates to that region of the well wall defined by the lowermost in use about 50p1 of sample volume of a SOOpl individual sample vessel.
24. A vessel as claimed in any preceding claim, wherein the vessel is formed by a single step injection moulding process
25. A vessel as claimed in any preceding claim, wherein the thermoplastic material is polypropylene.
26. A method of manufacturing a vessel comprising the steps of: a) forming a mould cavity with a first mould member and an opposing second mould member; b) providing at least one injection point and providing at least one outlet port; c) injecting into the at least one injection point a molten thermoplastic material of a volume larger than the volume of the mould cavity; d) allowing the excess molten thermoplastic material to exit the mould cavity from the at least one outlet port; e) allowing the molten thermoplastic material to set; f) removing the excess thermoplastic material that exited the mould cavity from the outlet port from the vessel formed in the mould cavity; wherein the first and second mould members form a vessel mould cavity such that the vessel formed will have at least one well for containing a sample, the well comprising an upper wall portion, an intermediate wall portion a lower wall portion and a bottom, wherein the wall thickness of the lower wall portion is less than the wall thickness of the intermediate wall portion and wherein the wall thickness of the lower wall portion is between about 0.06mm and about 0.17mm.
27. A method as claimed in Claim 26 wherein the vessel comprises a vessel as claimed in any of claims I to 25
28. A vessel substantially as herein described with reference to and as illustrated in any combination of the accompanying drawings.
29. A method substantially as herein described with reference to and as illustrated in any combination of the accompanying drawings.