NSB Amine Slide
Contents
1. e After hybridization it is recommended to wash the slides according to the below process Washing program for TECAN HS 400 model 1 Flow Washing buffer B at 37 C for 30 sec 2 Hold Washing buffer B at 37 C for 1 min 3 Repeat Steps 1 amp 2 4 Flow Washing buffer C at 25 C for 10 sec 5 Dry for 30 sec e Store the slides and protect them from light until scanning 3 2 2 Protocol for a microarray with long oligonucleotides 50 80mer using a Agilent microarray chamber kit G2534A e Make appropriate concentration of cDNA using Hybridization buffer B Typically the assay volume of target DNA solution is determined by the size of the hybridization chamber e Load denatured cDNA solution onto a gasket slide and carefully put an oligo microarray slide on top of the gasket slide e Keep the chamber in a hybridization oven and incubate at 40 65 C for 12 16 hour e After hybridization wash the slides with Washing buffer B at 37 C for 3 10 min followed by Washing buffer C for 10 sec and dry them using a nitrogen gas blower or a slide drier e Store the slides and protect them from light until scanning Note Please refer to Agilent microarray hybridization chamber user guide for detail direction of Agilent microarray chamber kit G2534A REFERENCE 1 V Sunkara B J Hong J W Park Sensitivity Enhancement of DNA microarray on Nanoscale Controlled Surface Biosensors and bioelectronics 22 7 1532 12. e Make appropriate concentration of target oligonucleotides or PCR products using Hybridization buffer A Typically the assay volume of target DNA solution is determined by the size of the hybridization chamber e Load denatured target solution onto a gasket slide and carefully put an oligo microarray slide on top of the gasket slide e Keep the chamber in a hybridization oven and incubate at 45 55 C for 1 min 4 hour e After hybridization wash the slides with Hybridization buffer A at 45 55 C for 3 10 min followed by Washing buffer A for 10 sec or D I water for 3 sec and dry them using a nitrogen gas blower or a slide drier e Store the slides and protect them from light until scanning Note Please refer to Agilent microarray hybridization chamber user guide for detail direction of Agilent microarray chamber kit G2534A 3 2 Gene expression profiling 3 2 1 Protocol for a microarray with long oligonucleotides 50 80mer in a hybridization machine like HS 400 TECAN e Make appropriate concentration of cDNA using Hybridization buffer B Typically the assay volume of target DNA solution is determined by the size of your hybridization chamber e Keep the slides in hybridization chambers of the machine insert cDNA solution denature cDNA at 90 C for 1 min and hybridize the slide at 40 65 C for 12 16 hour Denaturation function supplied by a hybridization machine can provide good hybridization performance
3. C and at a relative humidity between 50 and 80 e Incubate the printed arrays at 80 relative humidity in a humidity chamber or in the printing instrument kept at ambient temperature 20 25 C for over 3 hours Overnight incubation is recommended The humidity chamber can be made by using a saturated salt solution enclosed in an airtight container such as a glass desiccator jar A small glass dish or cotton wool can be used to hold the saturated salt solution at the bottom of the container e Place the slides in a slide holder and immerse in Washing buffer B Keep the buffer stirred at 37 C for 10 20 min Wash the slides with DI water Dry them using a nitrogen gas blower or a slide drier and store at 4 C Note The arrayed slides can be stored at 4 C under anhydrous environment for example under nitrogen environment or in a desiccator for at least 3 months before hybridization 3 HYBRIDIZATION It is well known that optimal hybridization and washing temperatures and times for DNA microarrays are dependent on the melting temperatures of DNA duplexes which are effected by salt concentrations and detergent or formamide amounts in buffer solutions Users are recommended to optimize the appropriate hybridization and washing conditions for the best DNA microarray results Standard examples for SNP genotyping and gene expression profiling are described below Prepare hybridization amp washing buffer Hybridization buffer A
4. for SNP genopyping NaCl 300 mM Na2xHPO 20 mM EDTA 2 0 mM and SDS 7 0 mM in sterile D I water pH 7 4 Hybridization buffer B for gene expression profiling 3 5X SSC and 0 30 SDS Washing buffer A 1X PBS Washing buffer B 2X SSC and 0 20 SDS Washing buffer C 0 2X SSC 3 1 SNP genotyping 3 1 1 Protocol for a microarray with short oligonucleotides 15 25mer using a hybridization machine HS 400 TECAN e Make appropriate concentration of target oligonucleotides or PCR products using Hybridization buffer A Typically the assay volume of target DNA solution is determined by the size of the hybridization chamber e Keep the slides in hybridization chambers of the machine insert target solution denature target DNAs at 90 C for 1 min and hybridize the slide at 45 55 C for 1 min 4 hour Denaturation function supplied by a hybridization machine can provide good hybridization performance e After hybridization it is recommended to wash the slides according to the below process Washing program for TECAN HS 400 model 1 Flow Hybridization buffer A at 45 55 C for 30 sec 2 Hold Hybridization buffer A at 45 55 C for 1 min 3 Repeat Steps 1 amp 2 4 Flow Washing buffer A for 10 sec 5 Dry for 30 sec e Store the slides and protect them from light until scanning 3 1 2 Protocol for a microarray with short oligonucleotides 15 25mer using a Agilent microarray chamber kit G2534A
5. 537 2007 2 S J Oh B J Hong K Y Choi and J W Park Surface Modification for DNA and Protein Microarrays OMICS A journal of Integrative Biology 10 3 327 343 2006 3 B J Hong V Sunkara J W Park DNA Microarrays on Nanoscale Controlled Surface with a Dendron Nucleic Acids Research 33 12 e106 2005 4 S J Oh J Ju B C Kim E Ko B J Hong J Park J W Park K Y Choi DNA Microarrays on a Dendron Modified Surface Improve Significantly the Detection of Single Nucleotide Variations in the p53 Gene Nucleic Acids Research 33 10 e90 2005 5 B J Hong S J Oh T O Youn S H Kwon J W Park Nanoscale Controlled Spacing Provides DNA microarrays with the SNP Discrimination Efficiency in Solution Phase Langmuir 21 4257 4261 2005 For detailed technical support or additional information please contact us Our special DNA microarray team can help you NSB POSTECH www nsbpostech com E mail support nsbpostech com or info nsbpostech com Phone 82 54 279 8416 Fax 82 54 279 8419
6. NSB Amine Slide USER MANUAL HANDLING AND STORAGE INSTRUCTIONS gt NSB Amine Slides as received can be stored in a desiccator at room temperature for one year After opening the package it is recommended that the slides be kept in a desiccator and used within six months for optimal outcome Use the slides in a clean environment since dust particles may cause problems Avoid direct contact with the surface while handling Hold the sides or edges of the slide and use only powder free gloves to minimize contamination and damage to the coating UG PRODUCT USE LIMITATIONS NSB slides are sold for research purposes only and are not intended for resale This product is not to be used in human diagnostics or for drug purposes in any way Certain arrays and or methods of preparation analysis or use may be covered by intellectual property rights in certain countries Use of this product is recommended only for applications for which the user has a license under proprietary rights of third parties or for technology for which a license is not required If you intend to use NSB slides for commercial purposes please contact us PROTOCOL 1 SLIDE ACTIVATION PROCESS Prepare reagents amp solvents Di N succinimidyl carbonate DSC Diisopropylethylamine DIPEA Anhydrous acetonitrile Dimethylformamide DMF Ethanol e Prepare 10 20 mM solution of di N succinimidyl carbonate DSC containing 0 1 v v DIPEA diisopr
7. opylethylamine in acetonitrile Ex 0 5 1g DSC in 200 ml acetonitrile and 0 2 ml DIPEA e Place the NSB Amine Slides in the above solution e Incubate it at room temperature r t for 4 hour Anhydrous environment is optional but recommended to protect the activated slide e Wash the slides with dimethylformamide DMF and then immerse them in DMF Stir the solution at r t for 20 mins or sonicate the solution at r t for 2 mins e Wash them briefly with ethanol dry using a nitrogen gas blower or a slide drier and keep under vacuum or under nitrogen atmosphere until spotting Storage The activated slides can be stored under vacuum at r t for a week and under nitrogen atmosphere for two days However it is recommended to activate the slides with DSC just before array fabrication 2 ARRAY FABRICATION Prepare spotting buffer amp washing buffer Spotting buffer 25 mM NaHCO and 5 0 mM MgCl in sterile D I water pH 8 5 10 DMSO Washing buffer B 2X SSC and 0 20 SDS Note Use of over 10 DMSO decreases the amounts of surface immobilized oligonucleotides leading to lower hybridization signal e Dissolve oligonucleotides to make 10 50 uM concentration in Spotting buffer Transfer the DNAs to 96 or 384 well microplate s 20 uM is recommended e Set up microarrayer and print slides according to the manufacturer s or laboratory protocol The printing environment should be dust free kept at temperature below 25
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