Sera-Mag Select Size Selection and PCR Clean-up reagent

Transcription

1 Sera-Mag Select Size Selection and PCR Clean-up reagent User Guide Product numbers:

2 Page Finder 1. INTRODUCTION 2 2. GENERAL TIPS FOR PROTOCOLS 2 3. MODES OF OPERATION Recovery Mode Single Size (Left Sided) Selection Mode Single Size (Right Sided) Selection Mode DUAL Size Selection Mode 5 4. FAQS AND TIPS 6 5. TROUBLESHOOTING 7 6. QUICK PROTOCOLS Single-round Binding Methods Two-round Binding Methods 8 7. RELATED PRODUCTS 9 8. LEGAL 9 1

3 1. INTRODUCTION About the user guide This guide provides information for use of Sera-Mag Select reagent for PCR reaction clean-up and DNA size selection in next generation sequencing (NGS) applications. Tips and FAQs are included to help the user to get the best results possible. A Troubleshooting guide is provided to help resolve specific issues that the user may encounter. Find your local support representative at 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. About Sera-Mag Select Sera-Mag Select reagent for PCR clean-up and size selection is based on the well-known Solid-Phase Reversible Immobilization technology, for selective binding of DNA fragments for applications such as NGS, PCR reaction cleanup, etc. It combines the ease of magnetic bead technology, using the exceptional binding characteristics of Sera-Mag Carboxyl Speedbeads with an optimised binding solution in a readyto-use formulation. The DNA size range isolated from the process can be tailored to suit the user s end requirements, by adjusting the amount of reagent that is mixed with a fixed volume of sample. As a general rule, the smaller the DNA fragment to be bound, the larger the volume of the reagent that should be added (relative to the initial sample volume). DNA of 100 bp size or greater can be reliably recovered using Sera-Mag Select. 2. GENERAL TIPS FOR PROTOCOLS For best results, Sera-Mag Select reagent should be stored longer-term at 2 8 C. However, it may be stored at ambient temperature for up to four months. Before use, it should be fully equilibrated to ambient temperature and homogeneous suspension of the magnetic beads, preferably via continuous mixing on rollers. Aliquots of Sera-Mag Select reagent are best dispensed by reverse pipetting for best reproducibility, as the reagent is somewhat viscous. Precision and accuracy of this pipetting are critical for best reproducibility of size selection results. Due to the viscosity, for manual dispensings, it is advisable to wipe off droplets of excess reagent adhering to the outside of the tip before dispensing into the sample tube, especially if using quantities at the lower end of the ratio range. As seen with many competitor products, aspiration of supernatants after magnetic separation may require care so as not to dislodge some beads from the magnet as the supernatant surface meniscus passes down over the bead pellet. This effect can be avoided by the appropriate choice of magnetic separators, or by addition of low levels of surfactants (for example TWEEN -20) in the input samples. 3. MODES OF OPERATION Sera-Mag Select size selection reagent can be used in a variety of modes of operation, depending on the desired outcome. MODE Description Upper size selection limit RECOVERY SINGLE SIZE (Left Sided) SELECTION SINGLE SIZE (Right Sided) SELECTION DUAL SIZE SELECTION Uses a single maximum volume aliquot of Sera- Mag Select reagent to bind all DNA above 100 bp within a sample Uses a single volume aliquot of Sera-Mag Select reagent, less than maximum, to bind DNA above a defined molecular weight cut off, which is eluted and retained. Uses two volume aliquots of Sera-Mag Select reagent: The first as for single (left sided) selection: beads with bound DNA fraction discarded; supernatant retained. The second as for recovery, adding additional reagent to bring total reagent addition up to recovery level, yielding DNA below a defined molecular weight cut off. Uses two volume aliquots of Sera-Mag Select reagent: The first as for single (right sided) selection: beads with bound DNA fraction discarded; supernatant retained. The second as for single (left sided) selection: adding additional reagent to bring total reagent addition to less than recovery level. Fixed at MAX = Fixed at MAX = Variable MAX Variable MAX Lower size selection limit Fixed at MIN Variable Min Fixed at MIN Variable Min Upper size selection limit at MAX = implies DNA effectively up to full-length genomic DNA. Lower size selection limit at MIN implies DNA of size less than ~100 bp 2

4 3.1. Recovery Mode This mode is typically used for PCR clean-up and DNA concentration. The reagent is applied at a single level to bind all DNA above 100 bp. This may include: After gel electrophoresis, a typical result is as shown in Figure 1: For maximum recovery, Sera-Mag Select should be added to 2.5 the sample volume. For example, if the intention is to recover purified PCR amplicon from a 50 μl volume PCR reaction, then the required volume of Sera-Mag Select is (2.5 x 50 μl) = 125 μl. Example protocol: Input sample = 50 μl volume PCR reaction in a 1.5 ml tube. BINDING / SEPARATION 1. Add in Sera-Mag Select reagent at 2.5X sample volume = ( μl) = 125 μl. 2. Vortex the binding mixture, then leave to incubate at ambient temperature for 10 minutes. Alternatively, mix for 10 minutes on a roller mixer set at 25 C for 10 minutes. 3. Briefly centrifuge the binding mixture, then place the tube in a magnet rack for 5 minutes to magnetically separate out the beads with bound PCR products. 4. Aspirate cleared supernatant liquors (containing unwanted PCR elements, including residual primers) to waste. WASHING BEADS 1. With the tube kept in the magnet rack, add in ~1 ml of wash solution (for example, ethanol 70% v/v aq.), in a manner so as not to unduly disturb the bead pellet. 2. Incubate for 1 minute, then aspirate the wash solution to waste. 3. Repeat the wash step once more. 4. Take care to aspirate as much wash solution as possible after the last wash. ELUTION OF BOUND DNA 1. Dry the bead pellet to remove all ethanol residue. For example, dry in a tube heater at 40 C for 10 minutes. 2. Resuspend the dried bead pellet in elution buffer (50 μl ), with vortexing. 3. Return the tube to the tube heater at 40 C for 15 minutes to complete extraction of the PCR products from the beads. 4. Vortex mix the tube until the beads have formed a homogeneous suspension. 5. Briefly centrifuge the mixture, then place the tube in a magnet rack for 5 minutes to magnetically separate out the beads. 6. Aspirate cleared supernatant solution containing the purified PCR products to a fresh tube. Fig. 1. PCR shows the crude mixture after amplification; the product amplicon band is evident at ~1350 bp. Residual oligonucleotide primers at <250 bp (enhanced here by spiking, for clarity); R shows the elution solution from the above protocol, which shows recovered PCR product amplicon, with no sign of primers. S shows the supernatant from the binding step (normally discarded), which shows the primers. There is minimal amplicon lost in this waste. Use of a volume of elution buffer that is significantly less than that of the input sample allows for product concentration as well as recovery; the amplicon band in R is then brighter than in the crude input as a result Single Size (Left Sided) Selection Mode In this mode, the reagent is applied at a single, intermediate level to bind DNA above a defined size cut off from the sample. The lower size cut off can be selected by adjusting the level of Sera-Mag Select added to the sample. As the level of applied reagent is increased from a value of around 0.4 (relative to the sample) upwards to the maximum level of around 2.5, large DNA fragments are recovered first; these are joined later by medium fragments and finally small fragments (Figures 2 & 3). Non-binding components are excluded at all applied levels of the reagent. The protocol is essentially the same as for the recovery mode. The only difference is that use of the reduced levels of reagent will cause otherwise binding DNA fragments at the lower end of the recoverable range to be rejected from binding to the beads, causing them to be left behind in the binding mix supernatant. Example protocol: Input sample = 100 μl volumes of a 100 bp dsdna ladder, diluted to an appropriate working stock level. 3

5 BINDING / SEPARATION 1. Add in Sera-Mag Select reagent at α sample volume (0.4 < α < 2.5) For example, if α = 0.8 is chosen, then the volume of reagent to add is: = [0.8 (initial sample volume)] = [0.8 (50 μl)] = 40 μl. The rest of the protocol, including all of the washing and DNA elution operations, is then exactly as for the recovery mode. Fig. 2. Left sided size selection DNA ladder was subjected to left sided size selection with given ratios of Sera-Mag Select to sample volume. Note that isolation of the largest fragment is not observed until a ratio of at least 0.45x is used. As the ratio is increased the other smaller DNA fragments are recovered in sequence until all fragments are recovered at ratio of ³ (greater than or equal to symbol) 2.0x. With this DNA ladder, isolation of the largest fragment is not observed until an X ratio of at least 0.45X is used, at which point the variable lower size cut off drops below the size of the largest sample fragment, allowing for its recovery. As the X ratio is increased beyond this level, the lower size cut off falls further; as a result the other smaller DNA fragments are recovered in sequence, until all are recovered at X ratios of 2.0. Left Sided Size Selection input Fig. 3. Left sided size selection Human genomic DNA was fragmented using NEBNext dsdna Fragmentase and 100 ng of fragmented DNA (50 µl in TE). Sample was subjected to left sided size selection with given ratios of Sera-Mag Select volume to sample volume. DNA was eluted in 50 µl TE and 1 µl was run on Bioanalyzer using Agilent High Sensitivity DNA Chip Single Size (Right Sided) Selection Mode This mode employs TWO sequential rounds of binding (Table 1): Round 1: equivalent to the single binding round used in Left Sided selection, except that it is the supernatant that is retained, not the bead fraction. This supernatant contains the inverse content of the Left Sided bead content displayed in Figure 3. It also contains all the non-binding components from the original sample, plus the non-bead components of the Sera-Mag Select size selection reagent. Round 2: equivalent to the single binding round used in recovery mode. A fresh aliquot of reagent is added to boost the concentration of binding agents to a level where all remaining bindable DNA will bind to the fresh beads. After magnetic separation, all the unwanted components (all the non-binding components from the original sample, plus the non-bead components of the Sera-Mag Select size selection reagent) are contained within the supernatant fraction, which is discarded. The fresh beads (to which the recovered DNA fragments are bound) are retained and processed by the standard wash and elution operations. For example, if a reagent level of 2.5 is considered for recovery mode and a reagent level of α is used for the initial binding; then the quantity of reagent added in the second binding round should be (2.5 -α). Example protocol: Input sample = 100 μl volumes of a 100 bp DNA ladder, diluted to an appropriate working stock level. BINDING / SEPARATION Round 1 1. Add in Sera-Mag Select reagent at α sample volume (0.4 < α < 2.5) For example, if α = 0.8 is chosen, then the volume of reagent to add = ( μl) = 80 μl. 2. Vortex the binding mixture, then leave to incubate at ambient temperature for 10 minutes. Alternatively, mix for 10 minutes on a roller mixer set at 25 C for 10 minutes. 3. Briefly centrifuge the binding mixture, then place the tube in a magnet rack for 5 minutes to magnetically separate out the beads with bound PCR products. 4. Aspirate cleared supernatant to a fresh tube. The original tube and its beads are discarded. BINDING / SEPARATION Round 2 1. Add in a fresh aliquot of Sera-Mag Select reagent. For example, if α = 0.8 was chosen for Round 1, then the volume of reagent to add for Round 2: = [( ) (initial sample volume)] = [(1.7) (100 μl)]= 170 μl. 2. Vortex the new binding mixture, then leave to incubate at ambient temperature for 10 minutes. Alternatively, mix for 10 minutes on a roller mixer set at 25 C for 10 minutes. 3. Briefly centrifuge the binding mixture, then place the tube in a magnet rack for 5 minutes to magnetically separate out the beads with bound PCR products. 4. Aspirate cleared supernatant to waste. The new tube and its fresh bead content are retained and progressed. The standard washing and elution operations are then performed to isolate the purified and size-selected DNA fraction. (Figures 4 and 5). 4

6 Tube ID Vol sample /µl Round 1 Round 2 X ratio Vol µl X ratio Vol µl Total reagent used X ratio Vol µl A X X X B X X X Right Sided Size Selection C X X X D X X X E X X X F X X X G X X X H X X X I X X X J X X X K X X X L X X X Table 1: Right sided size selection for ratios from 0.35x - 2.5x at round 1, including second round values for addition of reagent to achieve 2.5x ratio required for recovery mode. After gel electrophoresis analysis of the eluted samples, typical results for the two single-size selection modes are as shown in Figure 4. SINGLE SIZE (Right Sided) SELECTION Fig. 4. Right sided size selection DNA ladder was subjected to right sided size selection with a given ratios of SeraMag Select volume to sample volume. Note that right size selection is essentially the reverse of the left sided selection. At Round 1, at ratios of & (less than or equal to symbol) 0.40x all DNA fragments are recovered. As the ratio is increased the DNA fragments are lost in sequence from the largest down, until none are recovered at ratios of ³ (more than or equal to symbol) 2.0x Fig. 5. Right sided size selection Human genomic DNA was fragmented using NEBNext dsdna Fragmentase and 100 ng of fragmented DNA (50 µl in TE, 2 ng/ul). Sample was subjected to right sided size selection with given ratios of Sera-Mag Select volume to sample volume. DNA was eluted in 50 µl TE and 1 µl was run on Bioanalyzer using Agilent High Sensitivity DNA Chip Dual Size Selection Mode This mode uses a combination of both left sided and right sided size selection modes to purify a narrow range of DNA fragments centred around a desired size. In the first round, the amounts of Sera-Mag Select reagent are as for the first round of the single size (right sided) selection mode and defines the upper size limit. In the second round, left sided single size selection is carried out, adding sufficient Sera-Mag Select reagent to define the lower size limit. In this way, DNA fragments above and below the defined size are removed, leaving the desired fragments in the final extract. Example protocol: DNA fragmentation and size selection In the following example, lambda-dna was fragmented by a commercial DNA fragmentase kit, based on the supplier Instructions For Use. Conditions were chosen that gave a broad range of dsdna fragments between bp. Input samples = μl volumes from a stock of fragmented genomic-length lambda-dna. Experiment plan for addition of Sera-Mag Select reagent: Size range ID input Sample vol µl Round 1X ratio/vol µl Round 1X ratio / vol µl Total X ratio/vol µl HI X / 40 µl +0.10X / 10 µl 0.50X / 50 µl MED X / 50 µl +0.15X / 15 µl 0.65X / 65 µl LO X / 65 µl +0.35X / 35 µl 1.00X / 100 µl BINDING / SEPARATION Round 1 1. Add to the input samples the Round 1 aliquots of Sera-Mag Select reagent (one condition per sample). 2. Vortex the binding mixtures, then leave to incubate at ambient temperature for 10 minutes. Alternatively, mix for 10 minutes on a roller mixer set at 25 C for 10 minutes. 5

7 3. Briefly centrifuge the binding mixtures, then place the tubes in a magnet rack for 5 minutes to magnetically separate out the beads with bound PCR products. 4. Aspirate cleared supernatant liquors to fresh tubes. The original tubes and their beads are discarded. BINDING / SEPARATION Round 2 1. Add in the Round 2 aliquots of Sera-Mag Select reagent. 2. Vortex the new binding mixture, then leave to incubate at ambient temperature for 10 minutes. Alternatively, mix for 10 minutes on a roller mixer set at 25 C for 10 minutes. 3. Briefly centrifuge the binding mixture, then place the tube in a magnet rack for 5 minutes to magnetically separate out the beads. 4. Aspirate cleared supernatant liquors to waste. The new tube and its fresh bead content are retained and progressed. The standard washing and elution operations are then performed to isolate the purified and size-selected DNA fraction (Figure 6). Dual Sided Size Selection DS High (0.4 /0.5 ) DS Medium (0.5 /0.65 ) DS Low (0.65 /1 ) Fig. 6. Dual sided size selection Human genomic DNA was fragmented using NEBNext DNA Fragmentase and 100 ng of fragmented DNA (100 µl in TE) was subjected to dual sided size selection with given ratios of Sera-Mag Select volume to sample volume. DNA was eluted in 50 µl TE and 1 µl was run on Bioanalyzer using Agilent High Sensitivity DNA Chip. 4. FAQS AND TIPS 1. Can Sera-Mag Select magnetic particles be left in PCR reactions? No, ensure that no magnetic particles are carried over into the PCR reaction as this may impact downstream performance. 2. Can I use Sera-Mag Select for both clean up and size selection? Yes, Sera-Mag Select is manufactured to standards suitable for both PCR Clean-up and size selection for which, protocols are provided. 3. Will the size selection remove all primers and adapters from the PCR reaction? The effective elimination of both primers and adaptors from the PCR reaction can be accomplished by following the size selection protocol. 4. What is the smallest fragment I can select for in my reaction? The smallest fragment size we would recommend for size selection using Sera-Mag Select is 100 bp, please refer to the size selection protocol. 5. Can I use Sera-Mag Select to purify Genomic DNA? Sera-Mag select will bind genomic DNA, but is not designed for this purpose. Extremely large DNA strands can prove difficult to elute and may bind across magnetic particles causing them to aggregate. 6. Do I need to change program on my liquid handler if I am currently using AMPure XP beads? For the majority of applications, Sera-Mag Select should be substituted directly for AMPure XP without any change to the existing script(s), though we recommend that the use checks this before substituting into the workflow. 7. Is tighter size selection important? Selecting fragments in a wider range can reduce sequencing efficiency. Note that for a given amount of starting material, selection of a tighter size range from a fragmented genomic DNA sample will reduce overall yield, but will have a higher percentage of usable fragments which can improve amount of usable data from the sequencing run. Storage and Handling for Sera-Mag Select Speed-Beads 8. What are the proper storage conditions for the Sera- Mag Select? We recommend refrigeration between 2 C to 8 C when not in use, do not freeze. Store upright and keep bottle tightly sealed. 9. Can I leave Sera-Mag Select at room temperature and for how long? If the product is to be used regularly, it can be stored at ambient temperature for up to 4 months to avoid repeated temperature equilibration. 10. How long must I equilibrate at room temperature before using the Sera-Mag Select beads? Depending on the bottle size, this will be a minimum of 30 minutes at room temperature, for best performance ensure that the beads are fully equilibrated before use. If the product is to be used daily, it can be stored at ambient temperature for up to 4 months. 11. How long should I mix the Sera-Mag Select beads before adding them to my sample? Mixing time will be dependent on method used for mixing, but we recommend mixing until the solution is visibly homogeneous. 12. What is the best way to mix the Sera-Mag Select beads? Mix the beads on a roller mixer or by hand with gentle repeated inversion or gentle vortexing until they are visibly homogeneous. 13. Can the Sera-Mag Select be frozen? We do not recommend freezing as this is not necessary and may impact performance of the product. 6

8 5. TROUBLESHOOTING Issue Cause Solution No or very low DNA recovery i) Reagent is non-homogeneous: magnetic beads have settled out of suspension during shipment or storage. ii) Reagent has not been equilibrated to room temperature before use, after retrieval from chilled storage. Ensure reagent is fully homogenized and equilibrated before use. Roller mix for at least 1 hour (smaller units) or overnight (larger units) before use. Reduced or inconsistent DNA recovery i) Loss of magnetic beads during workflow incomplete settling to magnets. i) Ensure samples are settled fully with magnetic separator before aspiration of supernatant liquors up to 5 minutes may be required for this, depending on magnetic separator and process format. ii) When conducting wash steps, add wash solution away from the bead pellet to avoid unduly disturbing it. If this does happen, or is unavoidable due to process format, ensure beads are given time to re-settle fully (up to 5 minutes) before aspiration of wash solution. Inconsistent size selection profile ii) Loss of magnetic beads during workflow beads not sticking to magnets: some beads pulled off pellet as supernatant meniscus passes over during aspiration. Poor precision of sample and/or reagent volumes. Size selection profile is very sensitive to relative volumes at higher size cut offs. i) Magnetic separator is inadequate. Try an alternative device. ii) Include a surfactant such as TWEEN-20 with input sample, to reduce surface tension in the supernatant liquors. i) Ensure liquid dispenser (eg pipette) is properly calibrated. ii) Use reverse pipetting action to counter viscosity of the reagent. iii) For manual operations, ensure excess reagent that may adhere to the outside of the pipette tip is removed by wiping before dispensing. DNA recoveries decreasing over time Multiple use actions, leading to elevated risk of reagent degradation via contamination. i) Avoid long-term storage at room temperature. ii) Use a smaller unit of reagent to limit the number of repeat uses. iii) Consider sub-aliquotting reagent into smaller, single-process amounts. Purity of recovered DNA is poor, or has poor 260/230 nm absorbance ratio. i) Recovered DNA is contaminated with ethanol. ii) Recovered DNA is contaminated with reagent non-bead components i) Ensure as much wash solution as possible is aspirated, before proceeding with elution ii) Ensure residual ethanol in the wet bead pellet is removed by drying before elution. i) Ensure binding mix suspension is settled to a single volume by centrifugation before magnetic settling i.e. no droplets sit on walls and lid of tube. ii) Ensure volume of wash solution employed is sufficient to contact all surfaces of the process vessel; increase if necessary. 7

9 6. QUICK PROTOCOLS 6.1. Single-round Binding Methods BINDING / SEPARATION 1. Add in Sera-Mag Select reagent at required level: Mode X-ratios to use Required reagent volume for 50 µl sample Recovery Max 2.5 X 125 µl Single (Left Sided) Selection X µl 2. Vortex mix, then incubate at ambient temperature for 10 minutes. 3. Briefly centrifuge the mixture, then place the tube in a magnet rack for 5 minutes to magnetically separate out the beads 4. Aspirate cleared supernatant liquors to waste. WASHING BEADS 5. With the tube kept in the magnet rack, add in 1 ml of wash solution in a manner so as not to unduly disturb the bead pellet. 6. I ncubate for 1 minute, then aspirate the wash solution to waste. 7. Repeat the wash step once more. 8. Take care to aspirate as much wash solution as possible after the last wash. ELUTION OF BOUND DNA 9. Dry the bead pellet to remove all ethanol residue. 10. Re-suspend the dried bead pellet in elution buffer, with vortexing. 11. Place the tube in a tube heater at 40 C for 15 minutes to complete the extraction of DNA from the beads. 12. Vortex mix until the beads have formed a homogeneous suspension. 13. Briefly centrifuge the mixture, then place the tube in a magnet rack for 5 minutes to magnetically separate out the beads. 14. Aspirate cleared supernatant solution containing the eluted DNA to a fresh tube Two-round Binding Methods BINDING / SEPARATION 1. Add in the Round 1 aliquots of Sera-Mag Select reagent at required level: Mode Single (Right Sided) Selection Double Selection X-ratios Round 1 X-ratios Round 2 Total Reagent α 1 α 2 α 1+α 2 = 2.5 α 1 α 2 α 1+α 2 < Vortex mix, then incubate at ambient temperature for 10 minutes. 3. Briefly centrifuge the mixture, then place the tube in a magnet rack for 5 minutes to magnetically separate out the beads 4. Aspirate cleared supernatant liquors to fresh tubes for onward processing. The original tubes and their beads are discarded. BINDING / SEPARATION Round 2 5. Add in the round 2 aliquots of Sera-Mag Select reagent at required level. 6. Vortex mix, then incubate at ambient temperature for 10 minutes. 7. Briefly centrifuge the mixture, then place the tube in a magnet rack for 5 minutes to magnetically separate out the beads. 8. Aspirate cleared supernatant liquors to waste. WASHING BEADS 9. With the tube kept in the magnet rack, add in 1 ml of wash solution in a manner so as not to unduly disturb the bead pellet. 10. Incubate for 1 minute, then aspirate the wash solution to waste. 11. Repeat the wash step once more. 12. Take care to aspirate as much wash solution as possible after the last wash. ELUTION OF BOUND DNA 13. Dry the bead pellet to remove all ethanol residue. 14. Re-suspend the dried bead pellet in elution buffer, with vortexing. 15. Place the tube in a tube heater at 40 C for 15 minutes to complete the extraction of DNA from the beads. 16. Vortex mix until the beads have formed a homogeneous suspension. 17. Briefly centrifuge the mixture, then place the tube in a magnet rack for 5 minutes to magnetically separate out the beads. 18. Aspirate cleared supernatant solution containing the eluted DNA to a fresh tube. 8

10 7. RELATED PRODUCTS Item Product Image MagRack MagRack Maxi LEGAL GE, imagination at work, GE Monogram, and Sera-Mag are trademarks of General Electric Company. TWEEN is a trademark of Croda Group of Companies. NEBNext and Fragmentase are trademarks of New England Biolabs, Inc. Agilent is a trademark of Agilent Technologies, Inc. AMPure is a trademark of Beckman Coulter, Inc. All other third party trademarks are the property of their respective owners General Electric Company All rights reserved. First published September 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 on request. Contact your local GE Healthcare representative for the most current information. For your local office contact information, visit GE Healthcare UK Limited Amersham Place Little Chalfont, Buckinghamshire, HP7 9NA, UK 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. 100 Results Way, Marlborough, MA USA GE Healthcare Japan Corporation Sanken Bldg , Hyakunincho, Shinjuku-ku, Tokyo , Japan imagination at work AA KA PD