There are two main changes in the automated procedure: 1 The hybridization buffer A contains only 7. The Hs. Centrifuge again for 5 min at 16,g. A Pattern of DNA samples on a 2.
After the labeling reaction, each probe labeled with Cy3 or Cy5 consists of a DNA smear with most fragments below bp. MW: molecular weight marker. The graph B displays the 1mm absorbance of each sample according to the light wavelength in nm. Test and reference genomic DNA show one single pick at nm, while each probe labeled with Cy3 or Cy5 shows a second pick with maximum absorbance at nm and nm, respectively.
The table C reports Cy3 or Cy5 incorporation rates for each labeled probe. For each spot, the log2ratio value is calculated, normalized by the median of all log2ratio values and plotted against the genome position of the corresponding clone. Some local log2ratio variations indicate the presence of small copy number changes between the 2 individuals examples indicated by red arrows.
The authors would like to thank Heike Fiegler who developed some of the methods described in this chapter. This work was supported by the Wellcome Trust. However, both molecules are very sensitive to environmental conditions such as light, high ozone and humidity levels The use of DNA samples which do not fulfill these quality criteria may result in failure of the labeling reaction or low quality of the array-CGH results i.
We have noticed that commercially available Cot-1 DNA tends to show batch to batch variations in terms of suppression efficiency. Suppressive hybridization with high quality Cot-1 DNA results in log2ratio values close to 0. The use of lower quality Cot-1 DNA may result to incomplete repeat suppression, compressed abnormal ratios and increased background ratio variability.
We routinely use the scanner from Agilent technologies: this fully-automated system with a slide loading carousel uses dynamic auto-focus to keep features in focus while scanning. Although spot intensity extraction programs are usually supplied with commercially available scanners, they can be purchased separately.
For further data analysis described in Chapter 3 , the ratio values between the fluorescent intensities in both channels on every spot on the array are calculated usually after the subtraction of local background fluorescence. Intensity ratios are then normalized, for example by dividing each individual ratio by the median ratio value of all clones. National Center for Biotechnology Information , U. Methods Mol Biol. Author manuscript; available in PMC May Copyright and License information Disclaimer.
Copyright notice. The publisher's final edited version of this article is available at Methods Mol Biol. See other articles in PMC that cite the published article. Abstract Array-CGH involves the comparison of a test to a reference genome using a microarray composed of target sequences with known chromosomal coordinates. Introduction Array-CGH was developed in the late nineties 1 , 2 to detect DNA copy number changes at high resolution along the genome or locus of interest see Chapter 3 for a general introduction on the method.
Materials 2. Cover slip. Hybridization chamber. Hybridization and detection, automated protocol Hs. PRO stations Tecan, Inc. Open in a separate window. Indeed, the said device has been shown to withstand pressures greater than about psi FIG. Preferably the panels are made of a polymer that has a higher melt temperature than the melt temperature of the overmolded material. These materials enable the over molding process to be more robust such that thermal heat transfer into the premolded material does not lead to thermal distortion of the premolded parts geometry, which compromises the device's overall form, fit, and function.
The over-molded jacket FIG. The jacket attaches the two panels together by a combination of thermal fusion of the thermoplastic materials and mechanical interlock of the panel's flanges 17 and flanges The jacket begins at the top of the sample holder 89 FIG.
The inner diameter of the jacket 89 FIG. A split parting line was used on the outer surface 91 FIG. Parting line misalignments usually occur when molds are designed to open in two halves. Eliminating the split-parting line on the inner diameter of the overmold helps ensure that undesirable gaps between the cap and the device do not occur.
The absence of these gaps help to ensure the acceptable form, fit and function of the fluid seal at the sample holder and vial cap.
The jacket includes an overmolded material that maintains the same diameter from the top of the sample holder FIG. The bottom edge of the jacket includes a integrated wide flange 88 FIG. This feature facilitates high speed spinning operations in a centrifuge by distributing the loads or stresses generated by the device into the shoulder of a receiver vial.
The side view shows that the width of the wide flange feature 88 FIG. The transition of the wide flange feature 88 FIG. The transition curves from the overmold seal are intentionally gradual so as to minimize stress concentrations. The outer diameter of the wide flange feature 88 is the same as the outer diameter at the top rim of the sample holder.
This feature enables the sample holder to be inverted and spun in the same receiver vial as shown in FIG. The wide flange 88 provides enough material support to keep the device in place during centrifugal spin operations that may be equal to and greater than 1, Gs. Experiments have shown that the flared feature enabled devices to be spun at centrifugal spin loads as high as 16, Gs for one hour without damage.
When the flared feature was not used, some devices plastically deformed and collapsed into the bottom of the receiver vial. Failure analysis of devices showed that the flared geometry was needed to keep the walls of the vial as circular as possible, and also distribute the contact stress between the device and the support rim 85 of the vials below the plastic yield stress of the vial. The wall thickness of the overmolded wall at the top of the device 89 FIG.
A suitable thickness is 0. When a vial cap 80 is pressed onto the sample holder to establish a liquid seal, hoop stresses are generated.
When devices are spun in a centrifuge at speeds that generate 16, Gs or more, the mass of the cap combined with the snap-fit feature create tensile-hoop stresses in the sample holder. If these stresses are high enough, the side wall of the sample holder fractures along the knit line. The knit line refers to the joint where the two or more melt flows of plastic meet and are fused together during the overmold process.
The overmolded jacket FIG. In order to ensure that devices do not fracture, the wall of the sample holder must be thick enough to prevent the: a elastic deformation that would enable caps to open—which is undesirable, and b plastic deformation and rupture that would allow leakage of the sample fluid—which is also undesirable.
For the low-protein-binding material of choice—styrene butadiene copolymer—a wall thickness of at least 0. When the wall thickness 89 FIG.
Sample volumes of less than 0. Having a device that has a sample volume up to 0. Care must be taken to ensure that any additional heat used does not cause the bottom of the panels to melt and collapse—which is undesirable. The two panels of the device need to be thick enough and stiff enough to support the overmold pressure at the nose of the panels and at the center of the panels. Experiments using current geometry and styrene-butadiene material revealed that a wall thickness of at least 0.
This thickness and a suitable wall strength was needed even though the panels were supported by a steel-core pin FIG. The surface of the pin that is closest to the membrane surface was relieved to ensure that the membrane never comes into contact with the core pin FIG. The retentive layer of the membranes can be damaged when the membrane comes into contact with core pins and can be scratched as the parts are ejected from the overmold.
To prevent membranes from being pulled away from panels and scratched by the surface of the core pin , the pin was fabricated with vent holes that enable air from the mold cavity to be evacuated through the center of the core pin.
This unique core pin design enables the overmolding of devices in a manner that does not over pressurize and blow the attached membrane off of the panels. Special attention must be given to the design of cooling ports in the overmolded cavity. In the most extreme cases the pre-molded panels deformed enough to completely close off the drain holes. In some cases the heat effects were sufficient to allow panels to slightly move away from the overmolded cavity and allow plastic leak and to flow over the exterior wall of the panel.
In some cases the leakage was small enough that the devices were still of good quality. In the worst cases the plastic flowed all the way up to the drain holes and partially filled them. This was considered to be undesirable because the flow through the drain holes was restricted. The adverse affects of heat on the pre-molded panel can be overcome by improving thermal cooling of the mold cavity and core pin, and by using a valve gate at the plastic injection port 92 FIGS.
Narrow edge gates create high levels of shear flow in the plastic, which generate more heat. These shear flows can be reduced by using a valve gate, which has a larger cross-sectional flow area.
This increased area reduces shear heating affects and enables the overmolded cavity to be more easily filled. Valve gates are using in injection molds to direct the flow of melted plastic polymer from the hot runner into the mold cavity. To achieve the best molding results, this flow of polymer should be directed towards a solid surface in the mold cavity such as a core pin 95 FIG. This flow needs to be broken up into turbulent swirls to prevent material flow marks and jetting, which could roughen the surface of finished parts.
The adverse affects of overmolding heat can also be overcome by placing cooling lines closer to the part's surface and by including thermal cooling lines in the core pin. This can usually be accomplished by using typical cooling fluids such as water or propylene glycol solutions. The adverse affects of heat can also be overcome by using mold inserts that have higher values of thermal conductivity.
Materials that have a higher thermal conductivity will enable heat to be drawn away from parts more effectively than when materials having a lower thermal conductivity are used. These inserts are usually fastened into the mold cavity, and help transfer heat away from a pre-mold more effectively that when one type of steel is used. Typically inserts can be made using metals, such as beryllium, copper and aluminum. This seal feature can be integrally molded into the panel's outer surface and functions like a mechanical O-Ring seal.
When the overmolded cavity is closed over the panels, the cavity wall makes intimate contact with the integrally-molded-seal feature. This seal helps prevent leakage of plastic into the drain holes 18 of the sample holder. Preferably the core pin FIG. Although using a core pin made of P20 steel, hardened to a Rockwell hardness of Rc, may be adequate to successfully make a small number of devices, the core pin may ultimately deform, which compromises the housing-burst strength of devices as measured by housing-burst pressure.
Accordingly, preferably the core-pin material is an H13 steel hardened to a Rockwell hardness of Rc, which is more durable. Thus, the material hardness and geometry of the core-pin design need to be carefully controlled to successfully make devices on a commercial scale. It is also very important to control the following factors during the overmolding process so as to ensure that a good device is made:.
To make good pre-molded panels, an injection mold valve gate needs to be positioned at the nose 92 FIG. Placing the valve at the nose of panels enables plastic material flow into the mold and preferentially positions the material knit lines in the underdrain structure, and not across the area where the membrane is attached.
Allowing the knit lines to occur at the membrane attachment site compromises the devices retention performance. Heating processes used to attach the membrane material can cause the knit lines to open in an undesirable way, which allows fluids to leak around the seal.
The membrane coupons are die-cut using an automated, matched die set in order to achieve the coupon-to-coupon dimensional accuracy that is needed. The process of automated die cutting, pick-and-placement, and heat sealing of coupons is very important to the manufacture of these devices.
Automated processes help reduce surface damage that can occur to the retentative layer of the membrane coupons. Automated processing also helps to reduce the labor content of manufacturing these devices compared to using manual manufacturing processes. The adverse effects of operator to operator variability are also reduced when automated manufacturing processes are used.
The top edge 89 FIG. The inner diameter 90 of the sample holder must be formed on one core pin FIG. This ensures that a good sealing qualities are achieved between the sample holder and the vial cap. Since the overmolded design requires that a split mold can be used, the parting line for the mold halves should be positioned 90 degrees away from the plastic knit lines. This design helps to prevent the alignment of residual molding stresses, knitlines, and parting lines.
This feature enables the samples holder to sustain higher stresses during centrifugal spinning. Higher stress capabilities enable higher spin speeds to be used, which enable shorter filtration times to be achieved.
These combined features provide a unique value proposition to customers. A labyrinth seal feature 19 and 20 FIG. As mentioned above, an exemplary labyrinth seal feature includes a raised geometry on one side of the panel's center line 20 FIG.
The shape of this feature is preferably symmetric about the center axis of the panels, which enables one mold cavity to make the two panels that will be assembles into one device. This helps reduce the cost of having to mold two separate panels to achieve the same assembly.
The seal feature also creates a tortuous path between the inner volume of two assembled panels and the outer space surrounding the panels. The tortuous path helps seal off the edge of the panels when the overmold is closed, which enable the overmold plastic to flow and seal the two panels together. This tortuous path helps prevent the overmold plastic from flowing into the inner volume of the sample holder. The shape and location of the drain holes 18 on the panels were specifically designed to help achieve the low variations in dead-stop volumes, acceptable fluid flow, and acceptable mold durability.
The core pins used to form the drain holes were designed with a 5 degree draft on each side. This draft improves the pin's strength and enable the pins to easily separate from the molded panels. The draft also creates a tapered hole, such that the more open side of the hole was placed on the inner portion of the panels. The protocol was kept as agents added organic solvent results represented on amicon ultra centrifugal filters protocol was measured.
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