Target Specifier 3 – User`s Manual
Contents
1. NI COMBIMATRIX CustomArray 90K Microarray Hybridization and Imaging Protocol PTLO20 OOK aus AAA Hybridization and Imaging of CustomArray 90K Workflow Target Preparation RNA or DNA user chooses kit Biotin or Fluorochrome Labeling Pre hybridization RNA Fragmentation RNA targets only 40 min 10 15 min hands on Hybridization 4 16 hours 10 min hands on Hybridization Washing 30 min Biotin labeled Targets Post hybridization Blocking Labeling and Washing 60 min 20 min hands on Fluorochrome labeled Targets Final Washing 15 min Imaging 10 min Protocol for CustomArray 90K Microarray Hybridization and Imaging PTLO20 Rev 00 Page www combimatrix com Hybridization and Imaging of CustomArray 90K Table of Contents TV OU COMEN sg acterssrita 5 shrcnec ae outages retinas in tates S arin aetna ogee can ees anaes aay eis Sere ii EFOCQUCTION sennae a r TA 1 Materials and EQuipment Provided cswivecnintavesounesornacaniddeianesiaunseaooladaes aa aa eea isane 1 Materials and Equipment Required not ProVided cccccccececseceeeeee sees sees sees eee ee esse ee eeeeeeeeeseeeeeaeeeeetataenenetes 1 CUSLOMAKaY SUK ASSCIMDIY ainin aE captain sane hue tanh acca anv A T ek eames daans bigs dae enon tcnta 3 Handling of Assembled Microarrays es ancacsiacaiaesnsancyiestoinawetiawedcsapmaenie2. Page 14 of 15 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 QO Production Efficiency Manufacture eight 90K custom arrays in less than 24 hours O Versatility Use the same design or different designs in a single run CustomArray Synthesizer Specifications Min Max Oligo Length Up to 50 mer Min Max
3. 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 temperature 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 90K Microarray Hybridization and Imaging PTLO20 Rev 00 Page 7 of 15 www combimatrix com RNA Fragmentation RNA Target Samples Only IMPORTANT CombiMatrix recommends that RNA target samples be fragmented prior to hybridization I 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 F
4. edge will feel rougher than the glass surface 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 90K with LifterSlip coverslip Protocol for CustomArray 90K Microarray Hybridization and Imaging PTLO20 Rev 00 Page 12 of 15 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 CustomArray microarray for subsequent re hybridization using the CombiMatrix CustomArray Stripping Kit see Appendix A Do not allow
5. 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 90K 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 for CustomArray 90K Microarray Hybridization and Imaging PTLO20 Rev 00 Page 4 of 15 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 12X 10 Tween 20 100 ul 0 1 0 5M EDTA 560 ul 40mM Nuclease free water Total Volume 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 Tabl
6. 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 90K Microarray Hybridization and Imaging PTLO20 Rev 00 Page 11 of 15 www combimatrix com Imaging of CustomArray 90K IMPORTANT The CustomArray 90K must be scanned wet using the I maging Solution supplied NOTE The LifterSlip coverslip provided with the CustomArray has been specifically designed to retain the I maging Solution without contacting the microarray surface Do not use a standard coverslip with CustomArray 90K 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 Remove the PBS Wash Solution from the hybridization chamber Carefully remove the hybridization chamber from the microarray by removing the clips and lifting the Hybridization Cap off the slide surface Immediately cover the semiconductor microarray surface with the Imaging Solution Using thin tipped forceps pick up a fresh LifterSlip and hold it so that the raised edges face the microarray The raised edges can be detected by gently rubbing an edge with the tip of the forceps the raised
7. Feature Capacity for Array 2 240 12 544 94 500 Microarray Format 1 x 3 CustomArray slide m O m O Production Capacity One to eight slides per run Protocol for CustomArray 90K Microarray Hybridization and Imaging PTL020 Rev 00 Page 15 of 15 www combimatrix com
8. K Microarray Hybridization and Imaging PTLO20 Rev 00 Page 9 of 15 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 6X SSPET Wash solution rinse the hybridization chamber fill the chamber cover the portals with adhesi
9. aseneaiasaniuamecamesemas petakonaad ma eeeannes 4 Preparation TOF IY ONIGIZael OM assets ex a E nad si tican wesana Suu a acantiuanecateanauseionaetorns 5 SOIWTIONS REGUIFCO TOF All Targets srera ida tacos aninwieds chara venues AE ees inoe es 5 Solutions Required Only for Biotin labeled Targets cccccececeeeeeseeeeeeeeeeeeeeeeeeeeeeeeeeteseneneeteeenennas 6 PFA CLA OV acana sahs etter Saba latte etn cca od nara age estate eta ace ee ahs aaa la cidatasactaa oats 7 RNA Fragmentation RNA Target Samples Only cccccccccscececeeeeeteeeeeee ease eeeeeeeeeeeteteeeeeeaeaeaeaeeeeeretenetetenetatataeags 8 YOZA BV Raph tact a Bes tad psa eyes cerned sare via apalncraetsceasacel octet acacia nga E ane Moines sean n ae eadcnee pate 9 FAY DIGI ZATION WaS NNO eo a woes tart acai taieatad E a a ya Galea die gue Aaland Guan pata lana ae aa 10 Post hybridization Labeling for Biotin labeled SAMPI S cccccececeeeeseeeeeeeeeaeeeeeeeeereeseaeaeaeeueuesaueeeeretetetatatatags 11 Final Washia a iusee teins tunataaga vistas vay on Tus aay hats au ei a A a aentee ea saeeteal ae ee 11 Mading OF CUSEOMAL Fay QO ronrisrin daiar a aE dia cndlacmade nitrate seng ech tea taaeaalta due E T N 12 Appendix A Related Products Available from COMbIMAtrix cccccccecscceceeeeeeeeeeeeeeeaeeeeeaeeeeeeeeaeaeeetatasaueetaeannenes 14 CombiMatrix Rotisserie Holders for CustomArray MicroarrayS cceeeeeeeeeeeeeeeeeeeeeeeeeeeteeeeneeeeeeeeegs 14 CombiMatrix Custo
10. e 2 Wash Solutions 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 ul 10 Tween 20 9 7 ml Nuclease free water 2 mi 10X PBS PBST Wash 2X PBS 0 1 Tween 20 100 ul 10 Tween 20 7 9 ml Nuclease free water 2 ml 10X PBS 8 ml Nuclease free water PBS Wash 2X PBS Protocol for CustomArray 90K Microarray Hybridization and Imaging PTLO20 Rev 00 Page 5 of 15 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 NOTE You can replace the Biotin Blocking Solut
11. ion with the 5X PBS Casein Blocking Buffer for both blocking and labeling steps Protocol for CustomArray 90K Microarray Hybridization and Imaging PTLO20 Rev 00 Page 6 of 15 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 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 Hybridization Solution Hybridization Temperature C 25 Formamide 45 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 120 ul Volume Cap Final Concentration 2X Hyb Solution Stock 6X SSPE 0 05 Tween 20 20mM EDTA Nuclease free water 50X Denhardt s solution 5X Salmon sperm DNA 10mg ml 1 ul 100 ng pl 1 SDS 0 05 Total Volume 120 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
12. ling Solution using the 1 mg ml stock solution of Fluorolink Cy5 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 2 Using the PBS Wash solution rinse the hybridization chamber fill the chamber and incubate the microarray at room temperature for
13. mArray Stripping Kit ANd Clamp ccc ccc ceccee seen cece eee eeeeeeeeeeeeeeeeeeeeeeeeeeeeenegs 14 Appendix B CombiMatrix CustomArray SYNthe SiZer cccccecececeeseeeeeeeeeueueueeeereteeeeaeaeaeeeeeesauaueeeeretetatatataeags 15 Protocol for CustomArray 90K Microarray Hybridization and Imaging PTLO20 Rev 00 Page ii www combimatrix com Hybridization and Imaging Protocol Introduction This manual describes how to hybridize labeled target samples to the CombiMatrix CustomArray 90K microarray and how to prepare the microarray for imaging DNA or RNA target samples can be labeled by incorporation of either biotin or a red channel fluorochrome Cy5 or AlexaFluor 647 fluorescent dyes Preparation of labeled targets is the user s responsibility Hybridization is performed with 2 to 8 ug of labeled target sample per microarray 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 90K must be performed using a high resolution fluorescent scanner that is compatible with a 1 x3 slide format with a minimum resolution of 5um and an adjustable focus The hybridized CustomArray 90K can be stripped of targets using the CombiMatrix Stripping Kit see Appendix A and then re used according to
14. mmended 2 to 8 ug per sample Salmon sperm DNA 10mg ml 100 ng ul 1 SDS a 0 04 Nuclease free water to 120 ul Total Volume 120 ul a Replace formamide with water when working with the labeled DNA targets e Salmon sperm DNA solution should be heat denatured as for preparation of the pre hybridization solution 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 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 90K can be attached to standard rotisseries using holders available from CombiMatrix see Appendix A Protocol for CustomArray 90
15. of 15 www combimatrix com O 10 mg ml Sonicated Salmon Sperm DNA sheared Ambion Cat 9680 O 1 SDS O 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 O Deionized DI Formamide a RNA Fragmentation Reagents see the section RNA fragmentation For Biotin labeled targets Q E Acetylated Bovine Serum Albumin BSA 20 mg ml solution Ambion Cat 2614 or 5X PBS Casein Blocking Buffer BioFX Laboratories Cat PBSC 0100 01 Biotin detection reagent Fluorolink Cy5 labeled streptavidin GE Healthcare Amersham Biosciences Cat PA45001 Protocol for CustomArray 90K Microarray Hybridization and Imaging PTLO20 Rev 00 Page 2 of 15 www combimatrix com CustomArray 90K Assembly IMPORTANT The Hybridization Cap and Clips are intended for single use only Dispose of these items upon completion of this protocol 1 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 2 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 Semiconductor Stop Clip Figure 1 CustomArray 90K microar
16. r shaft is 610012 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 a 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 90K microarrays to be hybridized at a time CombiMatrix CustomArray Stripping Kit and Clamp QO CombiMatrix CustomArray Stripping Kit for 90K Product Number to be determined 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 a 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 CustomArray 90K Microarray Hybridization and Imaging PTLO20 Rev 00
17. ragmentation 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 Final Concentration 1 M Tris Acetate pH 8 1 adjust pH with glacial acetic acid Se ae 200 mM KOAc 0 49 g 500 mM MgO Ac osg 150 mM Water To 10 ml Total volume 2 Set up the RNA fragmentation reaction see Table 7 Use 2 to 8 ug of the labeled RNA for each CustomArray 90K microarray Table 7 RNA Fragmentation Reaction Reagent Volume for 120 ul Hybridization Cap Nuclease free water to 19 2 ul 5X RNA Fragmentation Solution 4 8 ul Labeled RNA 2 8 ug per microarray Total Volume 24 ul 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 90K Microarray Hybridization and Imaging PTLO20 Rev 00 Page 8 of 15 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 120 ul Volume Cap Final Concentration 2X Hyb Solution Stock 6X SSPE 0 05 Tween 20 20mM EDTA DI Formamide for RNA targets only 25 Labeled targets DNA or fragmented RNA Varies up to 24 pl 20 80 ng ul reco
18. ray Hybridization Cap and Clips Solution Portals Figure 2 Assembled CustomArray 90K microarray with Hybridization Cap and Clips Protocol for CustomArray 90K Microarray Hybridization and Imaging PTLO20 Rev 00 Page 3 of 15 www combimatrix com Handling of Assembled Microarrays l 2 4 Wear gloves at all times when handling microarrays and reagents After a CustomArray 90K microarray is assembled and re hydrated keep the Hybridization Cap in place during all hybridization and wash steps 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 90K 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
19. 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 90K Microarray Hybridization and Imaging PTLO20 Rev 00 Page 13 of 15 www combimatrix com Appendix A Related Products Available from CombiMatrix CombiMatrix Rotisserie Holders for CustomArray Microarrays To improve microarray performance use of a rotisserie oven or a rotating incubator is recommended to ensure mixing during hybridization CustomArray 90K microarrays can be attached to standard rotisseries using the same holders that are available from CombiMatrix for CustomArray 12K microarrays QO 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 QO CombiMatrix 8 CustomArray Rotisserie for 12K Product Number for 3 8 diamete
20. the hybridization protocol described in this manual Materials and Equipment Provided IMPORTANT Do not touch the semiconductor microarray surface Wear gloves when handling CustomArray 90K microarrays can be stored in a cool dry place for up to 4 months CustomArray 90K microarray Hybridization Cap 120 ul Volume Clips for Hybridization Cap LifterSlip coverslip Imaging Solution DOUCUOD Materials and Equipment Required not provided Target DNA or RNA samples labeled with biotin or fluorochrome dyes Rotisserie oven CombiMatrix CustomArray Holder for rotisserie oven optional see Appendix A 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 95 C Heating Block Micropipettors and nuclease free pipette tips Sterile plastic ware Gloves powder free 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 Nuclease free water 50X Denhardt s solution 0 5 M EDTA pH 8 0 10 Tween 20 20x SSPE Buffer DUOUOD DoUcCDO DUOUCOD Protocol for CustomArray 90K Microarray Hybridization and Imaging PTLO20 Rev 00 Page 1
21. ve 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 Ifthe 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 90K Microarray Hybridization and Imaging PTLO20 Rev 00 Page 10 of 15 www combimatrix com Post hybridization Labeling for Biotin labeled Samples NOTE If working with fluorochrome labeled targets skip this step 1 Prepare the Dye Labe
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