CustomArray™ 12K Microarray
Contents
1. 1 Pre heat an incubator to 65 C 2 Seta hybridization rotisserie oven to the desired hybridization temperature See Table 4 for recommended hybridization temperatures Table 4 Recommended Hybridization Temperatures Sample Hybridization Solution Hybridization Temperature C RNA 25 Formamide 45 DNA No Formamide 50 IMPORTANT The hybridization protocol has been optimized for standard oligonucleotide lengths 35 40mers with a balanced melting temperature Tm of 722C If non standard oligonucleotide lengths are used conditions such as hybridization temperature and hybridization buffer composition must be optimized empirically 3 Prepare fresh Pre hybridization Solution see Table 5 Table 5 Pre hybridization Solution Reagent For 100 ul Volume Cap Final Concentration 2X Hyb Solution Stock 50 pl 6X SSPE 0 05 Tween 20 20mM EDTA Nuclease free water 34 ul 50X Denhardt s solution 10 ul 5X Salmon sperm DNA 10mg ml 1 pl 100 ng ul 1 SDS 5 pl 0 05 Total Volume 100 ul Heat the Salmon sperm DNA solution to 95 C for at least 5 minutes and then place on ice for at least 1 minute before use 4 Fill the hybridization chamber with nuclease free water Avoid introducing air bubbles into the chamber Cover the solution portals with adhesive tape to prevent evaporation Incubate at 65 C for 10 minutes Remove the microarray from the incubator and bring to room tempe2. Replace formamide with water when working with the labeled DNA targets P Salmon sperm DNA solution should be heat denatured as for preparation of the pre hybridization solution 2 Denature the Hybridization Solution at 95 C for 3 minutes and then cool for 1 minute on ice Spin down the solution in a microcentrifuge for 5 seconds at maximum speed to collect condensate Remove the adhesive tape from the microarray and pipet the Pre hybridization Solution out of the hybridization chamber 5 Fill the hybridization chamber with the Hybridization Solution and mix gently with repeated pipetting A small air bubble can be introduced to improve the mixing process if the microarray is rotated during hybridization NOTE Usually the volume of Hybridization Cap is slightly less than 100 ul You can adjust the final volume of Hybridization Solution to 95 instead of 100 ul if you want the whole amount to fit into the chamber 6 Carefully wipe excess solution from the surface of the Hybridization Cap with a lint free tissue and cover the solution portals with adhesive tape 7 Load the microarray onto the rotisserie in the hybridization oven and incubate at the desired hybridization temperature for 4 16 hours with gentle rotation NOTE To improve microarray performance use of a rotisserie oven or a rotating incubator is recommended to ensure mixing during hybridization The CustomArray 12K can be attached to standard rotisseries using
3. M Tris Acetate pH 8 1 adjust pH with glacial acetic acid 2 mi 200 mM KOAc 0 49 y 500 mM MgOAc 0 32 g 150 mM Water To 10 ml Total volume 10 ml 2 Set up the RNA fragmentation reaction see Table 7 Use 1 to 4 ug of the labeled RNA for each CustomArray 12K microarray Table 7 RNA Fragmentation Reaction Reagent Volume for 100 ul Hybridization Cap Nuclease free water to 16 ul 5X RNA Fragmentation Solution 4 ul Labeled RNA 1 4 ug per microarray Total Volume 20 pl 3 Incubate at 95 C for 20 minutes Place on ice 4 Add the entire Fragmentation Reaction volume to the Hybridization Solution Protocol for CustomArray 12K Microarray Hybridization and Imaging PTLOO6 Rev 01 Page 7 of 14 www combimatrix com Hybridization 1 Prepare the Hybridization Solution see Table 8 IMPORTANT The hybridization buffer composition depends on the type of nucleic acids used as targets For RNA but not DNA targets it should include 25 formamide Table 8 Hybridization Solution Reagent For 100 ul Volume Cap Final Concentration 2X Hyb Solution Stock 50 ul 6X SSPE 0 05 Tween 20 20mM EDTA DI Formamide for RNA targets only 25 ul 25 Labeled targets DNA or fragmented RNA 1 to 4 ug per sample Varies up to 20 ul 10 40 ng ul recommended Salmon sperm DNA 1 Omg ml 1 yl 100 ng pl 1 SDS 4 ul 0 04 Nuclease free water to 100 ul Total Volume 100 pl
4. dyes Q Rotisserie oven a CombiMatrix CustomArray Holder for rotisserie oven optional see Appendix A Q Standard high resolution fluorescent microarray scanner CombiMatrix recommends the Axon Instruments Genepix 4000B and 4200A microarray scanners or the Perkin Elmer ScanArray 4000 5000 Lite and Express microarray scanners a 952C Heating Block a Micropipettors and nuclease free pipette tips Q Sterile plastic ware Q Gloves powder free a Adhesive tape Scotch Brand Magic Transparent Tape is suitable for hybridization temperatures of 50 C or less for extended incubations at higher temperatures use a PCR sealing tape such as Nunc Brand PN 232702 clear polyolefin liner or PN 276014 aluminum liner a Nuclease free water a 50X Denhardt s solution a 0 5 M EDTA pH 8 0 Protocol for CustomArray 12K Microarray Hybridization and Imaging PTLOO6 Rev 01 Page 1 of 14 www combimatrix com Q 10 Tween 20 Q 20x SSPE Buffer a 10 mg ml Sonicated Salmon Sperm DNA sheared Ambion Cat 9680 a 1 SDS a 10X Phosphate buffered Saline 1 37M Sodium Chloride 0 027M Potassium Chloride 0 08M Sodium Phosphate dibasic 0 02M Sodium Phosphate monobasic pH 7 4 Ambion Cat 9625 For RNA targets Q Deionized DI Formamide a RNA Fragmentation Reagents see the section RNA fragmentation Q Q For Biotin labeled targets Acetylated Bovine Serum Albumin BSA 20 mg ml solution Ambion Cat 2614 o
5. holders available from CombiMatrix see Appendix A Protocol for CustomArray 12K Microarray Hybridization and Imaging PTLOO6 Rev 01 Page 8 of 14 www combimatrix com Hybridization Washing IMPORTANT Do not allow the microarray to become dry at any time Proceed rapidly when changing solutions Do not leave the hybridization chamber empty for any significant length of time NOTE 1 For every wash step we recommend rinsing the hybridization chamber with the corresponding Wash Solution prior to the wash incubation Add the Wash Solution to the chamber mix gently by pipetting remove the solution and fill the chamber again with the same solution NOTE 2 If the microarray has been hybridized with fluorochrome labeled targets protect it from light by covering with aluminum foil during all incubations exceeding 5 minutes NOTE 3 With the exception of the first wash step microarrays should be incubated in the Hybridization Wash solutions for a minimum of 1 minute However if processing multiple microarrays you can extend the wash incubation time until you rinse and fill all hybridization chambers 1 Prior to starting the wash procedure preheat the 6X SSPET Wash Solution to the hybridization temperature 45 or 50 C depending on the sample hybridized 2 Remove the microarray from the hybridization oven Remove the adhesive tape and pipet the Hybridization Solution out of the chamber 3 Using the pre heated
6. ml 10X PBS PBS Wash 2X PBS 8 ml Nuclease free water Protocol for CustomArray 12K Microarray Hybridization and Imaging PTLOO6 Rev 01 Page 4 of 14 www combimatrix com Solutions Required Only for Biotin labeled Targets NOTE _ If working with fluorochrome labeled targets skip this step 1 Prepare the 1 mg ml stock solution of the Fluorolink Cy5 labeled streptavidin This reagent is supplied as a lyophilized powder Dissolve in 1 0 ml of nuclease free water dispense into 10 20 ul aliquots freeze and store at 20 C Avoid repeated freeze thaw cycles 2 Prepare the Biotin Blocking Solution see Table 3 All reagents should be nuclease free The prepared Biotin Blocking Solution should be filter sterilized and can be stored at 4 C for up to 1 month Table 3 Biotin Blocking Solution Reagent Volume for 1 ml Final Concentration 10X PBS 200 ul 2X 10 Tween 20 10 ul 0 1 BSA 20 mg ml 500 ul 1 Nuclease free water 290 ul Total Volume 1 mi NOTE You can replace the Biotin Blocking Solution with the 5X PBS Casein Blocking Buffer for both blocking and labeling steps Protocol for CustomArray 12K Microarray Hybridization and Imaging PTLOO6 Rev 01 Page 5 of 14 www combimatrix com Pre Hybridization NOTE The CustomArray microarray must be re hydrated prior to hybridization A pre hybridization step is then recommended to block non specific binding of targets
7. 6X SSPET Wash solution rinse the hybridization chamber fill the chamber cover the portals with adhesive tape and return the microarray to the hybridization oven for 5 minutes with gentle rotation Remove the 6X SSPET Wash Solution from the hybridization chamber 4 Using the 3X SSPET Wash solution rinse the hybridization chamber fill the chamber and incubate the microarray at room temperature for 1 minute see NOTE 3 Remove the 3X SSPET Wash Solution from the hybridization chamber 5 Using the 0 5X SSPET Wash solution rinse the hybridization chamber fill the chamber and incubate the microarray at room temperature for 1 minute Remove the 0 5X SSPET Wash Solution from the hybridization chamber 6 Using the PBST Wash solution rinse the hybridization chamber fill the chamber and incubate the microarray at room temperature for 1 minute Remove the PBST Wash Solution from the hybridization chamber 7 If the microarray has been hybridized with fluorochrome labeled target samples proceed to the final wash step 8 If the microarray has been hybridized with biotin labeled target samples proceed to the Post hybridization Labeling Step Retain the PBST Wash Solution in the hybridization chamber until you are ready to proceed to the Post hybridization Blocking step Protocol for CustomArray 12K Microarray Hybridization and Imaging PTLOO6 Rev 01 Page 9 of 14 www combimatrix com Post hybridization Labeling for Biotin label
8. A AS COMBIMATRIX CustomArray 12K Microarray Hybridization and Imaging Protocol PTLO06 Hybridization and Imaging of CustomArray 12K Workflow Target Preparation RNA or DNA user chooses kit 1 3 days Biotin or Fluorochrome Labeling l Pre hybridization 40 min 10 15 min RNA Fragmentation RNA targets only pangs on dias 4 16 hours ybridization 10 min hands on Hybridization Washing 30 min Biotin labeled Targets 60 min Post hybridization Blocking 20 min ela Labeling and Washing hands on i Final Washing 15 min Imaging 10 min Protocol for CustomArray 12K Microarray Hybridization and Imaging PTLOO6 Rev 01 Page i www combimatrix com Hybridization and Imaging of CustomArray 12K Table of Contents Table of Contents sx ckic da entree eae a oi AR er he ate eth eet tee ji EPO UN et e etaa A a aa a Aaa E aTa ie 1 Materials and Equipment Provided cE eter ieee nnneneeeeees 1 Materials and Equipment Required not provided cn eeeeeees 1 CustomArray 1 2K ASSembly oiee alive natet aiid Gua Son Eia A a a aa neuen a a 2 Handling of Assembled Microarrays 0 0 2 2 tnnt rnrn ttt ES EENENNENERASEEEEENENEEEENEEEENEEEEEENEEEEEEEEEEEEEEEEEEEEEE 3 Preparation for HY rIdi Zati N monro aran a aeaa a 4 Solutions Required for All TargetS oooooocccccccoccncnncconnnnnnn
9. Array 12K Microarray Hybridization and Imaging PTLOO6 Rev 01 Page 13 of 14 www combimatrix com Appendix B CombiMatrix CustomArray Synthesizer The CustomArray Synthesizer enables researchers to make custom microarrays to their exact specifications While fulfilling their existing microarray requirements researchers can take microarrays to new frontiers by exploring unique and novel applications The platform consists of the CustomArray DNA Synthesizer instrument and freely programmable CustomArray microarrays The CustomArray technology utilizes a modified semiconductor adapted for biological applications The integrated circuits built into CustomArray contain arrays of microelectrodes that are individually addressable using embedded logic circuitry on the chip Placed in a specially designed fluidic chamber the chip under direction from software digitally directs the molecular assembly of oligonucleotides During this process a chip can rapidly synthesize several thousand different oligonucleotide probes in parallel each above a distinct electrode Additionally the platform utilizes standard phosphoramidite chemistry and the resultant DNA microarray slides can be read on common scanners CustomArray Synthesizer Product Number 610002 Q Production Efficiency Manufacture eight 12K custom arrays in less than 24 hours Q Versatility Use the same design or different designs in a single run CustomArray Synthesize
10. Do NOT allow the semiconductor microarray surface to become dry at any step in the protocol To add or remove solutions hold the microarray with the assembled Hybridization Cap at a 45 degree angle Use a 200 ul micropipettor with nuclease free pipet tips to add or remove solutions through the lower solution portal of the Hybridization Cap see Fig 3 Figure 3 Pipetting of solutions into the hybridization chamber of the assembled CustomArray 12K microarray Proceed rapidly when changing solutions during the pre hybridization hybridization and washing steps Do not leave the hybridization chamber empty for any significant length of time For all incubations longer than 10 minutes seal the solution portals of Hybridization Cap with non permeable adhesive tape to prevent evaporation Use Scotch Brand Magic Transparent Tape for hybridization temperatures of 50 C or less for higher temperatures use a PCR sealing tape such as Nunc Brand PN 232702 clear polyolefin liner or PN 276014 aluminum liner Wipe the surface clean with a lint free tissue before sealing If you plan to strip and re use a hybridized CustomArray 12K microarray see Appendix A do not allow the semiconductor surface to dry as this will prevent any further use Avoid prolonged storage of hybridized microarrays prior to stripping instead strip the microarrays then store them wet in Imaging Solution or 1X PBS at 4 C for a maximum of 2 weeks Protocol
11. ed Samples NOTE If working with fluorochrome labeled targets skip this step 1 Prepare the Dye Labeling Solution using the 1 mg ml stock solution of Fluorolink Cy56 labeled streptavidin Thaw an aliquot and make a 1 1000 dilution v v 1 ul per 1 ml in the Biotin Blocking Solution or 5X PBS Casein Blocking Buffer We recommend that you discard any unused Cy5 labeled streptavidin NOTE Prepare the Dye Labeling Solution fresh each time you hybridize microarrays 2 Remove the PBST Wash Solution from the hybridization chamber 3 Add the Biotin Blocking Solution or 5X PBS Casein Blocking Buffer to the hybridization chamber mix gently by pipetting and incubate the microarray at room temperature for 15 minutes Remove the solution from the hybridization chamber 4 Add the Dye Labeling Solution to the hybridization chamber mix gently by pipetting and cover the solution portals with adhesive tape Incubate the microarray at room temperature for 30 minutes Protect from light by covering with aluminum foil Remove the Dye Labeling Solution from the hybridization chamber 5 Using the PBST Wash solution rinse the hybridization chamber fill the chamber and incubate the microarray at room temperature for 1 minute Remove the PBST Wash Solution from the hybridization chamber 6 Repeat the PBST washing one more time Final Washing Remove the PBST Wash Solution from the hybridization chamber Using the PBS Wash solutio
12. edges face the microarray The raised edges can be detected by gently rubbing an edge with the tip of the forceps the raised edge will feel rougher than the glass surface 5 Lay the LifterSlip at an angle onto the microarray so that it is centered over the semiconductor area see Fig 4 First touch the Imaging Solution with one side of the LifterSlip then slowly lower the slip down taking care not to introduce air bubbles If bubbles still form lift one side of the LifterSlip with forceps or a razor blade to let the bubbles out and lower it down again Figure 4 CustomArray 12K with LifterSlip coverslip Protocol for CustomArray 12K Microarray Hybridization and Imaging PTLOO6 Rev 01 Page 11 of 14 www combimatrix com Carefully remove any excess Imaging Solution from the edge of the LifterSlip using a lint free tissue until it is resting evenly over the microarray Load the CustomArray microarray into the scanner taking care not to disturb the LifterSlip coverslip Follow the manufacturer s recommendations for loading the slide into the scanner After you complete the scan save the image as a tiff image file The data can be extracted from the image using the CombiMatrix Microarray Imager Software Please refer to the Quick Start Guide or the Microarray Imager User s Manual on our web site https webapps combimatrix com After imaging is completed you can proceed to stripping of the CustomAr
13. for CustomArray 12K Microarray Hybridization and Imaging PTLOO6 Rev 01 Page 3 of 14 www combimatrix com Preparation for Hybridization The following solutions can be prepared beforehand All reagents used should be RNase and DNase free Solutions Required for All Targets 1 Prepare the 2X Hyb Solution Stock see Table 1 This stock will be used for preparation of the Pre hybridization and Hybridization Solutions The 2X Hyb Solution Stock should be filter sterilized and can be stored at room temperature for up to 6 months Table 1 2X Hyb Solution Stock Reagent Volume for 10 ml Final Concentration 20X SSPE 6 ml 12X 10 Tween 20 100 yl 0 1 0 5M EDTA 560 ul 40mM Nuclease free water 3 34 ml Total Volume 10 ml Final concentration includes the EDTA from the SSPE 2 Prepare the Wash Solutions see Table 2 The prepared solutions should be filter sterilized and can be stored at room temperature for up to 6 months Table 2 Wash Solutions Step Solution For 10 ml 3 ml 20X SSPE 6X SSPET Wash 6X SSPE 0 05 Tween 20 50 ul 10 Tween 20 6 95 ml Nuclease free water 1 5 ml 20X SSPE 3X SSPET Wash 3X SSPE 0 05 Tween 20 50 ul 10 Tween 20 8 45 ml Nuclease free water 250 ul 20X SSPE 0 5X SSPET Wash 0 5X SSPE 0 05 Tween 20 50 pl 10 Tween 20 9 7 ml Nuclease free water 2 ml 10X PBS PBST Wash 2X PBS 0 1 Tween 20 100 ul 10 Tween 20 7 9 ml Nuclease free water 2
14. n rinse the hybridization chamber fill the chamber and incubate the microarray at room temperature for 1 minute Remove the PBS Wash Solution from the hybridization chamber Repeat a second time 3 Retain the PBS Wash Solution in the hybridization chamber until you are ready to proceed to the Imaging step Protocol for CustomArray 12K Microarray Hybridization and Imaging PTLOO6 Rev 01 Page 10 of 14 www combimatrix com Imaging of CustomArray 12K IMPORTANT The CustomArray 12K must be scanned wet using the I maging Solution supplied The LifterSlip coverslip provided with the CustomArray has been specifically designed to retain the maging Solution without contacting the microarray surface Do not use a standard coverslip with CustomArray 12K NOTE The Imaging Solution contains phosphate buffer which may precipitate during shipping If a precipitate is visible heat the Imaging Solution at 60 702C for about 5 minutes until it dissolves Allow the solution to cool to room temperature before applying it to the microarray 1 Remove the PBS Wash Solution from the hybridization chamber 2 Carefully remove the hybridization chamber from the microarray by removing the clips and lifting the Hybridization Cap off the slide surface 3 Immediately cover the semiconductor microarray surface with the Imaging Solution 4 Using thin tipped forceps pick up a fresh LifterSlip and hold it so that the raised
15. nd 647 fluorescent dyes Preparation of labeled targets is the user s responsibility Hybridization is performed with 1 to 4 ug of labeled target sample per microarray If two labeled samples are to be analyzed on the same array for a dual color experiment use 1 to 4 ug of each sample for a total of 2 8 ug The hybridization conditions recommended in this protocol are designed for gene expression analysis For other microarray applications please refer to the Combimatrix Website www combimatrix com or contact customer support support combimatrix com Imaging of the CustomArray 12K must be performed using a high resolution fluorescent scanner that is compatible with a 1 x3 slide format with a minimum resolution of 54m and an adjustable focus The hybridized CustomArray 12K can be stripped of targets using the CombiMatrix Stripping Kit see Appendix A and then re used according to the hybridization protocol described in this manual Materials and Equipment Provided IMPORTANT Do not touch the semiconductor microarray surface Wear gloves when handling CustomArray 12K microarrays can be stored in a cool dry place for up to 4 months CustomArray 12K microarray Hybridization Cap 100 ul Volume Gasket O ring Clips for Hybridization Cap LifterSlip coverslip Imaging Solution OoOooocvco Materials and Equipment Required not provided Q Target DNA or RNA samples labeled with biotin or fluorochrome
16. nncancnnnnnnnnnnnra da aK aa Kaa a a ciapa 4 Solutions Required Only for Biotin labeled Targets cccece cece cece eee eee eee ee eee ene nee eee need 5 Pre lt HYDIGIZAtON fh cdta a AA e li bat aaa a 6 RNA Fragmentation RNA Target Samples Only 0c nora nnnnnnnnannnnannnnnnnnnrcnnnnannn 7 HyDridizatiO Mesa eae eset i iv fly al oe ine ein teats advent Badenian Sia 8 Hybridization Washi Os coi e 9 Post hybridization Labeling for Biotin labeled SAMpleS ec terre eee nnnncrrnnanancnn 10 Final WASHING ese ooe iO iii 10 maging of C stomArray TK a ta 11 Appendix A Related Products Available from CombiMatriX ooononccnnnnnccnnococccnnnnnnnnnncoroncn eee nn nn rn nr nn 13 CombiMatrix Rotisserie Holders for CustomArray 12K MicrOarrayS oooococccocconconnncnnconncnnnnancnnnnannnnnns 13 CombiMatrix CustomArray Stripping Kit and Clamp c cece cece eee eee ee ee nena eae 13 Appendix B CombiMatrix CustomArray SynthesiZer 0 ci reer nnnannc 14 Protocol for CustomArray 12K Microarray Hybridization and Imaging PTLOO6 Rev 01 Page ii www combimatrix com Hybridization and Imaging Protocol Introduction This manual describes how to hybridize labeled target samples to the CombiMatrix CustomArray 12K microarray and how to prepare the microarray for imaging DNA or RNA target samples can be labeled by incorporation of either biotin or a fluorochrome Cy3 Cy5 or AlexaFluor 555 a
17. r 5X PBS Casein Blocking Buffer BioFX Laboratories Cat PBSC 0100 01 Biotin detection reagent Fluorolink Cy5 labeled streptavidin GE Healthcare Amersham Biosciences Cat PA45001 CustomArray 12K Assembly IMPORTANT The Hybridization Cap Clips and O ring are intended for single use only 1 Insert the provided Gasket O ring in the groove of the Hybridization Cap 2 Align the Hybridization Cap over the slide so that the top edge of the slide is flush against the stop on the Hybridization Cap and the Cap is centered over the semiconductor area 3 Secure the Hybridization Cap in place with the Clips provided see Fig 1 and 2 The Clips can be attached only if the Hybridization Cap is positioned with the top edge of the slide at the stop of the Cap Gasket O ring a Hybridization Cap CustomArray 12K Figure 1 CustomArray 12K microarray Hybridization Cap and Clips Protocol for CustomArray 12K Microarray Hybridization and Imaging PTLOO6 Rev 01 Page 2 of 14 www combimatrix com Solution Portals Figure 2 Assembled CustomArray 12K microarray with Hybridization Cap and Clips ak aN nn anun J Handling of Assembled Microarrays 1 2 4 5 Wear gloves at all times when handling microarrays and reagents After a CustomArray 12K microarray is assembled and re hydrated keep the Hybridization Cap in place during all hybridization and wash steps
18. r Specifications Min Max Oligo Length Up to 50 mer Min Max Feature Capacity for Array 2 240 12 544 Microarray Format 1 x 3 CustomArray slide Production Capacity One to eight slides per run Oooo Protocol for CustomArray 12K Microarray Hybridization and Imaging PTLOO6 Rev 01 Page 14 of 14 www combimatrix com
19. rature Remove the adhesive tape and aspirate the water out of the hybridization chamber 5 Fill the hybridization chamber with the Pre hybridization Solution Mix gently by pipetting A small air bubble can be introduced to improve the mixing process if the arrays are rotated during incubation Cover the solution portals with adhesive tape 6 Load the microarray onto the rotisserie in the hybridization oven and incubate at the desired hybridization temperature for 30 minutes with gentle rotation Protocol for CustomArray 12K Microarray Hybridization and Imaging PTLOO6 Rev 01 Page 6 of 14 www combimatrix com RNA Fragmentation RNA Target Samples Only IMPORTANT CombiMatrix recommends that RNA target samples be fragmented prior to hybridization n general 50 to 200 base fragments work best to maximize binding specificity and detection sensitivity This step can be completed during the Pre hybridization incubation NOTE Other established fragmentation protocols may be used in place of this protocol Alternatively the RNA Fragmentation Reagents from Ambion Cat 8740 can be used according to the manufacturer s protocol 1 Prepare the 5X RNA Fragmentation Solution see Table 6 The prepared solution should be filter sterilized and can be stored at room temperature for up to 6 months Table 6 5X RNA Fragmentation Solution RNA samples only Reagent Volume for 10 ml Final Concentration 1
20. ray microarray for subsequent re hybridization using the CombiMatrix CustomArray Stripping Kit see Appendix A Do not allow the semiconductor microarray surface to dry keep the microarray wet either in a tube or slide holder containing 1X PBS or in the Imaging Solution with the LifterSlip attached Avoid prolonged storage of hybridized microarrays prior to stripping instead first strip the microarray then store wet in Imaging Solution or 1X PBS at 4 C for a maximum of 2 weeks Protocol for CustomArray 12K Microarray Hybridization and Imaging PTLOO6 Rev 01 Page 12 of 14 www combimatrix com Appendix A Related Products Available from CombiMatrix CombiMatrix Rotisserie Holders for CustomArray 12K Microarrays To improve microarray performance use of a rotisserie oven or a rotating incubator is recommended to ensure mixing during hybridization CustomArray microarrays can be attached to standard rotisseries using holders available from CombiMatrix a CombiMatrix 32 CustomArray Rotisserie for 12K Product Number 610004 The 32 CustomArray Rotisserie for 12K allows the hybridization of 32 microarrays at a time The unit has four plates each with eight positions using ball detent clamps to secure the microarray The 32 CustomArray Rotisserie is supplied with a shaft that fits most Fisher Scientific sotemp Hybridization Incubators The Rotisserie can also be adapted to fit other incubation ovens Q CombiMa
21. trix 8 CustomArray Rotisserie for 12K e Product Number for 3 8 diameter shaft is 610012 e Product Number for 3 4 diameter shaft is 610013 The 8 CustomArray Rotisserie for 12K allows the hybridization of 8 microarrays at a time The Rotisserie will mount on a 3 8 or 3 4 diameter shaft of an incubation oven with simple spring clasps The model can also be adapted to fit other incubation ovens Please indicate the oven shaft diameter when ordering Q CombiMatrix 3 Tray CustomArray for 12K Product Number 610008 CustomArray 3 Tray for 12K is a three position tray that fits in place of the standard Affymetrix tray bucket in an Affymetrix oven As many as eight trays can be loaded in one oven allowing for 24 CustomArray 12K microarrays to be hybridized at a time CombiMatrix CustomArray Stripping Kit and Clamp a CombiMatrix CustomArray Stripping Kit Product Number 610011 The Stripping Kit enables re hybridization of a CustomArray microarray three times thus allowing the use of a single CustomArray up to four times Each kit contains reagents for 25 stripping reactions plus accessories for subsequent re hybridizations Q CombiMatrix CustomArray Stripping Clamp Product Number 610010 The CombiMatrix Stripping Clamp is designed to be used with the CombiMatrix CustomArray Stripping Kit The CustomArray Stripping Clamp is recommended for up to 100 stripping procedures Protocol for Custom
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