Amersham Cy3/HyPer5 reactive dye protocols for post labeling amino allyl-cdna and amino allyl-arna

Transcription

1 GE Healthcare Amersham Cy3/HyPer5 reactive dye protocols for post labeling amino allyl-cdna and amino allyl-arna For microarray applications Product booklet Codes:

2 Page finder 1. Legal 3 2. Handling Safety warnings and precautions Storage Expiry 6 3. Components 7 4. Description General precautions on using fluorescent dyes for microarray applications Protocols Preparation of aa-cdna or aa-arna Considerations for reactive dye coupling reactions Protocol 1 - Cy3 and HyPer5 reactive dye coupling to aa-cdna Protocol 2 - Cy3 and HyPer5 reactive dye coupling to amino allyl containing amplified RNA (aa-arna) Troubleshooting guide Appendix References Related products 25 2

3 1. Legal GE, imagination at work and GE monogram are trademarks of General Electric Company. Amersham, HyPer5, Cy, and CyDye are trademarks of GE Healthcare companies. All third party trademarks are the property of their respective owners. CyDye: this product or portions thereof is manufactured under an exclusive license from Carnegie Mellon University under US Patent Number 5,268,486 and equivalent patents in the US and other countries. The purchase of CyDye products includes a limited license to use the CyDye products for internal research and development, but not for any commercial purposes. A license to use the CyDye products for commercial purposes is subject to a separate license agreement with GE Healthcare. Commercial use shall include: 1. Sale, lease, license or other transfer of the material or any material derived or produced from it. 2. Sale, lease, license or other grant of rights to use this material or any material derived or produced from it. 3. Use of this material to perform services for a fee for third parties, including contract research and drug screening. If you require a commercial license to use this material and do not have one, return this material unopened to GE Heathcare Bio- Sciences, AB, Bjorkgatan 30, SE Uppsala, Sweden and any money paid for the material will be refunded. 3

4 General Electric Company All rights reserved. First published Dec All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available upon request. Contact your GE Healthcare representative for the most current information. GE Healthcare UK Limited. Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA UK. 4

5 2. Handling 2.1. Safety warnings and precautions Warning: For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals. All chemicals should be considered as potentially hazardous. We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing such as laboratory overalls, safety glasses and gloves. Care should be taken to avoid contact with skin or eyes. In the case of contact with skin or eyes, wash immediately with water. See material safety data sheet(s) and/or safety statement(s) for specific advice. Caution: These dyes are intensely colored and very reactive. Care should be exercised when handling the dye vial to avoid staining clothing, skin, and other items Storage Store at -15 C to -30 C. Do not use if desiccant capsule in foil bag is either pink or green. Please keep the dye vials in their original foil packaging with desiccant to prevent degradation of the reactive groups by environmental moisture. Once the foil packs have been opened, the Cy3 is highly susceptible to degradation even if stored in closely capped tubes. Tests have shown that the content of reactive groups decreases by about 1% per day when they are dispensed into aliquots and stored. Thus, stored aliquots have a very limited shelf life. 5

6 We recommend that foil packs of dye be opened only immediately prior to use Expiry For expiry date, please refer to outer packaging label. 6

7 3. Components Items are supplied individually packaged in foil packs. Twelve Cy 3 mono NHS ester pmol dye per vial Twelve HyPer5 mono NHS ester pmol dye per vial > 75% reactive dye per vial 7

8 4. Description Cy3 and HyPer5 dye Post-Labeling Reactive Dye Pack provides convenient, individually packaged, highly reactive Cy3 and HyPer5 mono functional NHS ester to label probes via the postlabeling (amino allyl) method for use in microarray applications. This method utilizes a chemical reaction to couple Cy3 and HyPer5 reactive dyes to synthesized cdna containing amino allyl groups (1). The post-labeling protocol consists of converting mrna into cdna in the presence of amino allyl dutp. After purification of synthesized cdna from unincorporated nucleotides (and degradation of the RNA template), Cy3 and HyPer5 reactive dyes are coupled to amino allyl cdna (aa-cdna) to generate probes for microarray hybridization. Note that each vial is sufficient for one labeling reaction. Hybridization of Cy3 and HyPer5 probes prepared using the postlabeling method yields bright signals on arrays and generates highly accurate data for gene expression profiling (2). HyPer5 reactive dye is resistant to degradation by light and ozone, an added benefit over Cy5. However, HyPer5 is not a direct replacement for Cy5 and users may need to optimize their protocols for use with HyPer5. HyPer5 is slightly red shifted compared to Cy5. HyPer5 absorbance maximum can be measured at 664 nm to more precisely determine dye incorporation levels. The individually packed Cy3 and HyPer5 reactive dyes are also suitable for making labeled probes from in vitro transcription products generated from Eberwine type (3) protocols utilizing amino allyl-containing amplified RNA (aa-arna). At GE Healthcare, we have developed two separate protocols to couple Cy3 and HyPer5 reactive dyes to both amino allyl containing cdna (aa-cdna) and amino allyl amplified RNA (aa-arna). In protocol 1, Cy3 and HyPer5 reactive dyes and aa-cdna are mixed 8

9 in 0.1 M sodium bicarbonate buffer (ph 9.0). Protocol 2 recommends DMSO solvent to resuspend Cy3 and HyPer5 reactive dyes for optimized aa-arna labeling. The labeling of CyDye and HyPer5 reactive dyes to both aa-cdna and aa-arna occurs efficiently under alkali conditions (ph ) which is achieved by using 0.1 M sodium bicarbonate buffer. It is important to make this buffer fresh for the labeling reactions. The recipe for 0.1 M sodium bicarbonate buffer is found in the Appendix (see page 23). To observe bright signals on microarrays, labeling density (dye ratio) in the range of nucleotides (one dye for every 20 to 100 unlabeled nucleotides) should be targeted for probes made by the post- labeling method. The labeling density can be controlled by titrating the amount of aa-cdna (or aa-arna) used in the coupling step. CyDyes are bright fluors with high extinction coefficients and as a consequence, do not require very high density cdna labeling for generating bright arrays. In fact, high density labeled cdna/ arna probes will eventually begin to reduce array signal due to CyDye quenching effects. For Cy3, it is recommended not to exceed a labeling density greater than 1 dye per every 15 nucleotides (dye ratios < 15) on microarray probes. The dye quenching effects for HyPer5 are minimal. For HyPer5, the total amount of dye incorporated per sample is more important than the specific nucleotide to dye ratio. HyPer5 couples very quickly (< 30 minutes) to amino allyl cdna and scientists at GE Healthcare are able to obtain > 30 pmoles of HyPer5 incorporated into the samples when starting with 20 µg total RNA templates. We have observed even better performance when starting with 1.0 µg mrna templates (nucleotide to dye ratios < 20 and > 70 pmoles HyPer5 incorporated per sample). We recommend hybridizing one entire labeling reaction per microarray (> 50 pmoles of HyPer5 labeled probes) to observe bright microarray signals. However, customer optimization may still be required depending on the type and quality 9

10 of starting templates used as well as the type and size of arrays hybridized General precautions on using fluorescent dyes for microarray applications Cy3 and HyPer5 dyes are sensitive to light and should be stored and used in the dark as much as possible. It is highly recommended to perform all Cy3 and HyPer5 microarray hybridization and washing steps in the dark to prevent signal loss from dye photo-bleaching. The microarray slides must be completely dried immediately after the final washing step of a hybridization protocol prior to laser scanning for image acquisition. Drying the slides in desiccator prior to scanning has been observed to increase HyPer5 signal. Wet arrays hybridized with fluorescent probes should have as little exposure to light as possible. Light exposure to arrays containing moisture or aqueous droplets will reduce signal during scanning. For scanners with fixed emission filters, Cy5 settings are recommended for scanning HyPer5. For scanners with adjustable filters, emission filters that capture light up to wavelengths of 710 nm are recommended. 10

11 5. Protocols 5.1. Preparation of aa-cdna or aa-arna Prior to performing Cy3 and HyPer5 reactive dye coupling, prepare aa-cdna or aa-arna according to standard methods. Purify the amino allyl-cdna or aa-arna away from unincorporated nucleotides, enzyme and buffer. When using CyScribe GFX purification columns, elute the aa-cdna or aa-arna with 0.1 M sodium bicarbonate buffer (ph 9.0). Quantify the cdna or arna using a spectrophotometer and measure absorbance at 260 nm. Proceed with recommended amount of amino allyl-containing cdna or arna template for Cy3 and HyPer5 reactive dye coupling reactions Considerations for reactive dye coupling reactions Tris-based buffers commonly used for eluting cdna or aa-arna at the final purification steps should be avoided since amines on Tris base compete with aa-cdna/aa-arna for dye coupling µg of aa-cdna or of aa-arna can be labeled from a single vial of Cy3 and HyPer5 reactive dyes. Ensure the reactive dye reaches ambient temperature before opening the vial (~ 10 minutes). After opening the vial, centrifuge at maximum speed to collect all dye particles at the bottom of the vial. The reactive dye coupling to amino allyl-containing cdna/arna template only occurs under alkali conditions between ph which is set using 0.1 M sodium bicarbonate buffer. Make this buffer fresh prior to setting up labeling reactions (see Appendix). 11

12 5.3. Protocol 1 - Cy3 and HyPer5 reactive dye coupling to aa-cdna Cy3 and HyPer5 reactive dyes have high solubility in aqueous buffers and can be readily dissolved by adding 0.1 M sodium bicarbonate buffer (ph ) directly to each vial. For coupling to aa-cdna, dissolving Cy3 and HyPer5 reactive dye with DMSO is not required. Maximal coupling of reactive dyes to aa-cdna template has been observed when both reactive dye and aa-cdna are mixed together in 0.1 M sodium bicarbonate buffer (ph ). 1. Spin dry the prepared amino allyl-cdna (aa-cdna) to ~ 5 µl using a SpeedVac with medium heat (or similar device). Do not over-dry to a pellet since cdna may be difficult to resuspend. 2. Bring the volume of aa-cdna to 40 µl by adding an appropriate volume of 0.1 M sodium bicarbonate buffer (ph ). Vortex to completely mix the aa-cdna. 3. Open a vial of Cy3 and/or HyPer5 reactive dye pouch after it has reached room temperature. Centrifuge the vial at maximum speed to bring contents to the bottom of the vial. 4. Add the entire 40 µl of aa-cdna (in 0.1 M sodium bicarbonate buffer) directly to a single vial of Cy3 and/or HyPer5 reactive dye. Vortex to resuspend the dye. 5. Briefly centrifuge at maximum speed to bring all contents to the bottom of the vial. 6. Incubate samples at room temperature for minutes in the dark to allow Cy3 and/or HyPer5 reactive dye to couple to aa-cdna. 7. To stop the coupling reaction, add 15 µl of 4 M hydroxylamine. 8. Incubate for 15 minutes at room temperature in the dark. 9. Proceed to purification of Cy3- and HyPer5-labeled cdna probes. Use CyScribe GFX Purification Kit (# ) for 12

13 Cy3- and HyPer5-probe purification. Quantify probe yield using a spectrophotometer. Take measurements at 260 nm to measure cdna yield and at 550 nm and 650 nm (or 664 nm) to quantify Cy3 and HyPer5 dye incorporation. 10. Store labeled probes in the dark at -20 C until needed for hybridization. Optimal microarray results are obtained if the labeled probes are used within a few days of preparation. Typical Cy3- and HyPer5 -cdna labeling data The following are examples of results obtained from two separate labeling reactions starting from 1 µg of mouse brain mrna and using a single vial of Cy3 and HyPer5 reactive dyes. The probes were purified with CyScribe GFX columns and yields quantified using a spectrophotometer. Probe RNA Template A 260 A 320 A 550 A 650 Probe Volume (ul) Cy3 mouse brain mrna ND 120 HyPer5 mouse brain mrna ND Calculations: cdna yield: for Cy3 = (A A 320 ) * 33 * dilution factor * sample volume = ( ) * 33 * 1 * 120 = ng for HyPer5 = (A 260 A 320 (0.25 * A 650 )) * 33 * dilution factor * sample volume = ( (0.25 * 0.088) * 33 * 1 * 120 = ng Note that 0.25 * A 650 is used as a correction factor for HyPer5 only since this dye does absorb at 260 nm. The HyPer5 dye is slightly red shifted compared to Cy5 with absorbance maximum at 664 nm. Researchers can measure absorbance at either 650 nm or more precisely at 664 nm to determine dye incorporation. 13

14 Probe yield: pmoles dye in sample = (A/E) * (1/W) * Z * df * 10 6 Where A = absorbance Cy3 at 550 nm or HyPer5 at 650 nm E = the extinction coefficient for Cy3 ( M- 1 cm- 1 ) or HyPer5 ( M- 1 cm- 1 ) Z = original sample volume expressed in microliters W = optical path of cuvette expressed in centimeters df = dilution factor For Cy3 = (0.054/ ) * (1/1) * 120 * 1 * 10 6 = 43.2 pmoles For HyPer5 = (0.088/ ) * (1/1) * 120 * 1 * 10 6 = 96.0 pmoles Nucleotide to dye ratio: cdna (in picograms) pmoles of dye incorporated Where the cdna average length is assumed to be 1000 bases and is the average molecular weight for a nucleotide in cdna. For Cy3 = ( ) = 46.6 For HyPer5 = ( ) = 19.1 The following table depicts typical results when HyPer5 NHS reactive dye was used in coupling reactions starting with 20 µg total RNA templates. Data shown are the averages from 35 HyPer5 labeling reactions. HyPer5 NHS reactive dye cdna yield ng Dye incorporation 98.9 pmoles Nucleotide per dye ratio

15 5.4. Protocol 2 - Cy3 and HyPer5 reactive dye coupling to amino allyl containing amplified RNA (aa-arna) The individually dispensed mono reactive dye packs can be used to label in vitro transcription products made from Eberwinetype reactions that incorporate amino allyl-utp into arna. Cy3 and HyPer5 reactive dye coupling to aa-arna is highly efficient when DMSO solvent (50%) is present during the labeling reaction. Typically, at least 2-fold higher yields are obtained from aa-arna labeling reactions when DMSO is included instead of 0.1 M sodium bicarbonate buffer (4). It is recommended to use the entire contents of Cy3 and HyPer5 reactive dyes for a single labeling reaction. As a guideline, 2 10 µg of amino allyl-incorporated arna (aa-arna) is a good starting amount to label from a single vial of Cy3 or HyPer5 reactive dye to make probes for hybridization to oligonucleotidebased arrays. The template (aa-arna) amount may need to be titrated to achieve desired labeling density, with optimal Cy3 ratios being for acquiring bright arrays. As mentioned previously, for HyPer5, the total amount of dye incorporated per sample is more important than the specific nucleotide to dye ratio since the dye quenching effects for HyPer5 are minimal. Customer optimization may be required when substituting HyPer5 for Cy5 in existing protocols. Warning! RNase free reagents and consumables are absolutely required to prevent degradation of RNA. 1. Spin dry the aa-arna to ~ 5 µl using a SpeedVac at medium heat setting, or similar device. Do not over-dry to a pellet since aa-arna will be difficult to resuspend. 2. Bring the volume of aa-arna to 20 µl by adding 0.1 M sodium bicarbonate buffer (ph ). Vortex to completely mix the 15

16 aa-arna template. At least 25% (v/v) of the reaction volume should contain 0.1 M sodium bicarbonate buffer. 3. Open Cy3 or HyPer5 reactive dye pouch after it has reached room temperature. Centrifuge at maximum speed to bring contents to bottom of the vial. 4. Add 20 µl of DMSO (Sigma D-8418) to each vial of Cy3 or HyPer5 reactive dye. Resuspend the dye by vortexing. Centrifuge to collect all contents to bottom of the vial. 5. Add the entire 20 µl of Cy3 or HyPer5 reactive dye (in DMSO) to aa-arna vial to bring the total volume to 40 µl. If less than 20 µl of Cy3 or HyPer5 reactive dye is used, make up the remaining volume with DMSO. The final DMSO concentration should be at least 50% in the reaction. Vortex to mix. Labeling reaction: aa-arna template 10 µl 0.1 M sodium bicarbonate buffer, ph µl Cy3 or HyPer5 reactive dye in DMSO* 20 µl Total Volume 40 µl *final DMSO concentration must be at least 50% in the labeling reaction. 6. Briefly centrifuge at maximum speed to bring all contents to the bottom of the vial. 7. Incubate at room temperature for minutes in the dark to allow Cy3 or HyPer5 to couple to aa-arna. 8. To stop the coupling reaction, add 15 µl of 4 M hydroxylamine. 9. Incubate for 15 minutes at room temperature in the dark. 10. Proceed to purification of Cy3- or HyPer5-aRNA probes using RNase-free reagents. Quantify probe yield using a spectrophotometer and take measurements at 260 nm for arna 16

17 yield and at 550 nm and 650 nm (or 664 nm for more precise HyPer5 measurement) for dye incorporation. 11. Store labeled probes in the dark at -70 C until needed for hybridization. Optimal microarray results will be achieved if labeled probes are used within a few days of preparation. Typical Cy3 and HyPer5 arna labeling data NHS reactive dyes can be used for labeling amino allyl modified amplified RNA (crna) synthesized by Eberwine amplification methods. The following table depicts typical results for arna labeling reactions starting from 500 ng of human universal RNA (Stratagene). Approximately 6 µg of amino allyl crna was generated and a small portion was used per dye coupling reaction. The probes were purified and yields quantified using a spectrophotometer. This labeling protocol is recommended for hybridization to oligonucleotide-based arrays. Amino allyl crna labeling with HyPer5 NHS reactive dye HyPer5 RNA probe yield 418 ng Dye incorporation 26.4 pmoles Nucleotide per dye ratio 47.0 Sample calculations: RNA probe yield: for Cy3 = (A A 320 ) * 37 * dilution factor * sample volume for HyPer5 = (A 260 A 320 (0.25 * A 650 ) * 37 * dilution factor * sample volume Note that 0.25 *A 650 is used as a correction factor for HyPer5 only since this dye does absorb at 260 nm. 17

18 The HyPer5 dye is slightly red shifted compared to Cy5 with absorbance maximum at 664 nm. Researchers can measure absorbance at either 650 nm or more precisely at 664 nm to determine dye incorporation. Probe Yield: pmoles dye in sample = (A/E) * (1/W) *Z *df * 10 6 Where A = absorbance Cy3 at 550 nm or HyPer5 at 650 nm E = the extinction coefficient for Cy3 ( M- 1 cm- 1 ) or HyPer5 ( M- 1 cm- 1 ) Z = original sample volume expressed in microliters W = optical path of cuvette expressed in centimeters df = dilution factor Nucleotide to dye ratio: cdna (in picograms) pmoles of dye incorporated Where the average probe length is assumed to be 1000 bases and is the average molecular weight for a nucleotide. 18

19 6. Troubleshooting guide Problem 1. Cy3 or HyPer5 probe labeling yield is poor Possible causes/solutions 1. Incorrect ph for the 0.1 M sodium bicarbonate buffer. The Cy3 and HyPer5 reactive dye coupling reaction occurs under alkali conditions between the ph ranges of Make fresh 0.1 M sodium bicarbonate buffer and ph to Now repeat the coupling reaction. 2. Labeled probe was lost during purification. Measure absorbance at 260 nm. If no cdna or arna was recovered, then the labeled probe was likely lost during purification. Ensure that the right concentration of ethanol was added to the wash buffer. Switch to CyScribe GFX Purification Kit for cdna probe purification. 3. Insufficient 0.1 M sodium bicarbonate buffer was present in the coupling reaction. For efficient coupling of Cy3 and HyPer5 reactive dyes to aa-cdna or aa-arna, at least 25% of reaction volume (v/v) must consist of buffer, otherwise, labeling yields may be inconsistent or low. 4. The amino allyl cdna synthesis reaction was poor. The Cy3 and HyPer5 reactive dyes couple to amine groups on cdna generated by incorporation of amino allyl- 19

20 Problem 1. Cy3 or HyPer5 probe labeling yield is poor (continued) Possible causes/solutions dutp during first strand cdna synthesis step. Quantify the aa-cdna after synthesis. If the cdna synthesis results in low yield, then fewer amino allyl groups will be present for dye couplings. Repeat the aa-cdna synthesis step, quantify the yield and repeat the reactive dye coupling reactions. Same applies when working with aa-arna template. Very low yield from in vitro transcription reaction will impact reactive dye coupling. 5. mrna or aa-arna degradation occurred. Analyze the cdna or arna probes on a denaturing 10% acrylamide gel to determine the size distribution which should be between Kb. If there are signs of degradation of RNA, then repeat in vitro transcription step. If the cdna distribution is small, then repeat aa-cdna preparation. 6. Tris-based buffers were used to wash and/or elute aa-cdna or aa-arna. This will cause quenching of the reactive dyes. During purification of aa-cdna, use 80% ethanol to wash the samples. If using the CyScribe GFX purification columns, elute the aa-cdna with 0.1 M sodium bicarbonate. 20

21 Problem 2. Template (cdna/ arna) yield dropped after labeling reaction 3. Low Cy3 or HyPer5 signals from array Possible causes/solutions 1. Reducing coupling volumes to less than 40 µl will impact cdna/arna recovery during the purification step. Increase the volume during the purification step. Increase coupling volume to recommended level of 40 µl. 1. Low dye incorporation into probes. Quantify the probe yield using a spectrophotometer and measure absorbance at 260 nm to quantify cdna/ arna yield. Measure absorbance at 550 nm and 650 nm (or 664 nm) for Cy3 and HyPer5. Use recommended amounts of probes for hybridization. 2. Dye ratios are too high. Make sure the labeling density is in the range of High dye ratios indicate incorporation was too low to observe bright signals on arrays. 3. Over labeling of probes. Over labeling can reduce signals due to dye quenching effects. High density labeled probes are also difficult to purify using standard resins leading to lower yields of cdna or arna for hybridization. 4. Hybridized slides were wet during scanning. The microarray slides must be completely dried after hybridization 21

22 Problem 3. Low Cy3 or HyPer5 signals from array (continued) Possible causes/solutions and washing steps prior to scanning to prevent signal loss from dye bleaching. Drying microarrays in a desiccator prior to scanning may increase HyPer5 signal. 5. Dye labeled samples exposed excessively to light which caused photobleaching of the dyes. Try to maintain samples and microarrays in dark containers shielded from ambient light. 22

23 7. Appendix Preparation of 0.1 M sodium bicarbonate buffer (ph ) 1. Add 4.2 g of NaHCO 3 to a one liter flask. 2. Add 500 ml of water and mix with a magnetic stirrer until the NaHCO 3 dissolves. The ph should be near Adjust the ph to by slowly adding ml solution of 0.1 M Na 2 CO 3 and monitoring the ph using a ph meter. 4. Filter-sterilize the buffer. Autoclave the buffer if working with aaarna. 23

24 8. References 1. DeRisi, J. L., Iyer, V.R., and Brown, P.O. Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278: , Richter, A., Schwager, C., Hentze, S., Ansorge, W., Hentz, M.W., Mukenthaler, M. Comparison of fluorescent tag DNA labelling method used for expression analysis by DNA microarrays. BioTechniques, Vol. 33, Number 3, , Eberwine, J., Yeh, H., Miyashiro, K., Cao, Y., Nair, S., Finnell, R., Zettel, M., and Coleman, P. Analysis of gene expression in single live neurons. Proc. Natl. Acad. Sci. 89: , Peter A. C. t Hoen, Floor de Kort, G. J. B. van Ommen and Johan T. den Dunnen. Fluorescent labelling of crna for microarray applications. Nucleic Acids Research, Vol. 31, No. 5, e20,

25 9. Related products Product Pack sizes Product code HyPer5 dctp 25 nmol HyPer5 dctp 250 nmol Cy3 dctp 25 nmol PA53021 Cy5 dctp 25 nmol PA55021 Cy3 dutp 25 nmol PA53022 Cy5 dutp 25 nmol PA55022 Cy3.5 dctp 25 nmol PA53521 Cy5.5 dctp 25 nmol PA55521 Cy3 dctp 250 nmol PA53031 Cy5 dctp 250 nmol PA55031 Cy3 dutp 250 nmol PA53032 Cy5 dutp 250 nmol PA55032 Cy3 dctp plus HyPer5 dctp Cy3 dctp plus Cy5 dctp Cy3 dutp plus Cy5 dutp 5 25 nmol 5 25 nmol 5 25 nmol 5 25 nmol 5 25 nmol 5 25 nmol PA55321 PA

26 Product Product code illustra CyScribe GFX Purification Kit purifications illustra CyScribe GFX Purification Kit purifications illustra GFX PCR DNA and Gel Band Purification Kit 100 purifications illustra GFX PCR DNA and Gel Band Purification Kit 250 purifications Ultrospec 3100 Pro with CyDye / 32 / 37/ 38 probe specific algorithm Microarray Hybridization Buffer RPK

27 27

28 GE Healthcare offices: GE Healthcare Bio-Sciences AB Björkgatan 30, Uppsala, Sweden GE Healthcare Europe GmbH Munzinger Strasse 5, D Freiburg, Germany GE Healthcare Bio-Sciences Corp. 800 Centennial Avenue, P.O. Box 1327, Piscataway, NJ , USA GE Healthcare Bio-Sciences KK Sanken Bldg , Hyakunincho, Shinjuku-ku, Tokyo , Japan GE Healthcare UK Limited Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA UK imagination at work PL Rev B