oparray user manual
Contents
1. Sample Spectrophotometer Reading Note i If performing the QC scan it is essential that all unconjugated dye is removed from the sample The QIAGEN PCR Purification Kit is sufficient for this If another kit is used for the last cleanup step insure that dye removal is sufficient so the QC scan doesn t detect dye carry over ii For dye conjugation DMSO does not interfere with the conjugation reaction Hence it is not necessary to dry down the dye aliquots if they are to be used directly Simply re suspend the cDNA in Carbonate buffer and add the dye in DMSO directly OpArray User Manual 11 Protocol for QIAGEN PCR Purification Kit e Add 35pl 100 mM NaOAc pH 5 2 to each reaction e Combine reactions in 1 tube e Add 500p PB Buffer e Apply sample to QlAquick column e Spin for 30 60 sec 13 000 RPM gt 10 000 x g e Discard flow through add 750pl PE buffer e Spin for 30 60 sec e Discard flow through e Spin for 1 min e Place column in new tube e Add 50pl EB Buffer or H O to membrane Note Eluted volume is typically around 48pl You may choose to elute with 1 2 x EB buffer and reduce the volume for hybridization in the spin vac e Spin for 1 min 10 OpArray User Manual IV Handling and Storage Conditions Slides should be handled with gloves to avoid smudges and dirt Store slides desiccated at 4 C in the absence of light Store oligo aliquots at 20 C V Reverse Transcription Labeling P2. Wash 1 1x SSC 0 03 SDS Wash 2 0 2 x SSC Wash 3 0 05 x SSC e Carefully remove the chamber and dry it with paper towels Note If you have several chambers to process dry them off and remove the screws but refrain from cracking them open until all the chambers are ready to open e Remove the slide from the chamber and place it into a slide rack to be washed e Gently plunge the rack up and down several times to get the cover slip to fall off the slide Wash for 2 3 min Note Lifter cover slips are reusable and should be washed in 70 EtOH prior to use e Always spin dry the slides for 5 min Note If using 50ml conical tubes to spin slides with the table top centrifuge be sure that the slide is orientated with the label side down OpArray User Manual 13 VI Hybridization Protocol A Probe Preparation Note The probe can be dried down to an appropriate volume Add SDS last just prior to denaturation to avoid precipitation Use 12 15pl probe mix per hybridization If using Lifter slips prepare 20pl of probe per hybrid ization Include Cot 1 DNA for Human Probe only Denature at 95 C for 2 min e Spin probe briefly Note This serves two purposes 1 to pellet condensation 2 to cool probe B Applying Probe e Align hybridization slide with reference slide Note Spots can be visualized by breathing on the slide to fog the surface e Place clean lifter cover slip Teflon side down over the array area
3. Note Lifter cover slips have Teflon edges along the two opposing outer edges This provides an advantage over traditional cover slips in that it allows for better diffusion of probe In addition air bubbles under the slip are more easily avoided e Place pipette tip along an open edge of the cover slip and slowly pipette the probe out of the tip Capillary action will draw the probe mix under the slip e Please note the hydration spot near the left side of slide A added last just prior to loading the chamber See figure 2 12 OpArray User Manual ll Kit Contents All OpArrays are shipped with the following materials e OpArray reference slide e Floppy disk containing the following files array layout gene list OpArray QC hybridization images Readme txt array parameters OpArray User Manual Ill Reagents and Equipment Needed Description Catalog OmniScript 200 units QIAGEN 205111 155 00 5 3 aminoallyl 2 deoxyuridine 5 triphosphate Sigma A0410 5mg for 277 40 Poly dA 18mer ae Custom Synthesis C C a EC Slide Chamber Several mao o NA pasm fa p a CC prices subject to change May 2001 6 OpArray User Manual QC Probe The quality of the probe may be checked by scanning it in a spectrophotometer 200 700nm Peak Sample 260nm cDNA 550nm Cy3 649nm Cy5 6 80 0 644 Si 0 4 g g 0 32 3 0 15 0 99 1 200 300 490 300 000 700 Wavelength Figure 1
4. OpArray User Manual Version 2 0 the ar 4 Genomics Trademarks Patent technology and or registered trademarks of Operon and QIAGEN OpArrays THE DNA COMPANY OP R2ON OPTs QlAquick is a trademark of QIAGEN OmniScript is a registered trademark of QIAGEN Registered names trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law Copyright 2001 Operon all rights reserved Operon Technologies Inc 1000 Atlantic Ave Alameda CA 94501 USA Ph 800 688 2248 510 865 8644 Email dna operon com Web www operon com Operon GmbH Nattermannallee 1 D 50829 K ln Germany Customer Service O0800 OP ORDERS 00800 67 67 33 77 Tech Support 00800 OP SUPPORT 00800 677 877 678 Email oligo operon com Web www operon com 2 OpArray User Manual Notes References These protocols are based on the following sources 1 The MGuide http cmgm stanford edu pbrown mguide 2 Eisen MB Brown PO DNA Arrays for Analysis of Gene Expres sion Methods in Enzymology 1999 303 179 205 3 DeRisi Lab Protocols http www microarrays org OpArray User Manual VIII Troubleshooting Lov yield cDNA Source RNA degraded Run RNA on an agarose gel to probe insure RNA integrity a Reagents not RNase free Run cDNA out on a gel RNA impure Check RNA purification procedure for errors Repurify RNA ee Nucleotides degraded Make frozen aliquots of nuc
5. bonate buffer pH 8 5 9 0 use Bicarbonate to adjust the pH Mix cDNA and Cy dye Incubate in the dark for 1 hr at room temperature E Quench Reaction Quench any un reacted Cy dye by adding primary amines Add 4 5pl 4M Hydroxylamine Incubate for 15 min in the dark at room temperature F Reaction Cleanup Il and Hyb Prep Cy3 and Cy5 reactions can be combined and cleaned up together or separately Removal of free dye can be performed with either gel filtration via spin column or Pasteur pipette or with various kits such as QIAGEN PCR Purification Kit The QIAGEN PCR Purifi cation Kit works well for this step However it is important to keep in mind that the DNA binding curve for silica on which this kit is based is favorable at low pH but falls off precipitously around pH 8 Thus it is essential that the pH of the reaction is below 7 5 before it at taches to the QIAGEN membrane OpArray User Manual
6. icroarrays has grown exponentially One widely used application for these gene chips has been the monitoring of gene expression In response to the demand for a reliable tool for this purpose Op eron has developed the OpArray a microarray containing op timized long oligonucleotide sequences Genes with a high degree of homology can present cross hybridiza tion problems that lead to false negatives and positives Currently available genomic primer sets provide oligos that are used in the amplification of DNA products spanning the full open reading frame ORF from start to stop codon In contrast Operon designs and synthesizes oligos representing a 7Ont region of the gene of inter est These sequence optimized 7Omers or OPTs are bioinformatically designed to minimize cross hybridization In addition sequences are Tm normalized and selected to minimize secondary structure 4 OpArray User Manual Slide A Slide B Figure 2 Applying the Probe e Lifter slips make pipetting the probe very easy It wicks under the cover slip with no bubbles slide B Note New cover slips are longer and should be placed on the slide 90 to the one shown in the picture above e Place the slide into the hybridization chamber e Pipette 15pl 3 x SSC near one of the edges of the slide away from the cover slip e Tighten the screws for the chamber cover and place the assem bly in a 67 C water bath for 6 12 hrs VII Post Treatment
7. leotides Inactive reverse Retry reaction with fresh enzyme transcriptase Failure to neutralize cDNA Add sodium acetate to sample prior to QlAquick following treatment with purification hydroxyalamine Cy Dye pack inactive Store dried Cy aliquots from DMSO in the absence of light at 4 C in a desiccator Inefficient dye incorporation pH of Carbonate buffer not Test pH of 100mM carbonate equal to 8 5 9 0 buffer Poor probe quality Take a spectrophotometer reading High slide of the probe prior to hybridization background See low yield cDNA probe inefficient dye incorporation Insufficient blocking of the Insufficient washing following slide hybridization can result in spots containing comet tails or smeary spots This can be avoided by thoroughly dunking slides during these washes Poor probe quality Take a spectrophotometer reading of the probe prior to hybridization See low yield cDNA Very low spot probe inefficient dye incorporation intensities 14 OpArray User Manual VI VII VIII Table of Contents Product Description Kit Contents Reagents and Equipment Needed Handling and Storage Conditions Reverse Transcription Labeling Protocol Hybridization Protocol Post Treatment Troubleshooting OpArray User Manual 12 13 I Product Description In the past few years as genome project sequencing information has become widely available the popularity and usefulness of DNA m
8. rotocol This protocol utilizes Mono Reactive Cyanine dyes to label cDNA after reverse transcription Incorporation of a nucleotide containing an alkyl amino group allows post RT conjugation Although there are other labeling methods available we have found this protocol to be simple and effective Aliquotting 50X dNTP Mix ee eon oe dATP 500 pM 100mM 10pl dCTP 500 pM 100mM 10pl dGTP 500 pM 25mm 100mM 10pl Dye Packs e Resuspend Cy dyes in 72 pl DMSO e Aliquot 4 5 pl to 16 tubes e lLyophilize in Speed Vac e Store sample in the absence of light at 4 C in a desiccator OpArray User Manual 7 A Reverse Transcription Reaction Note 2pg of mRNA should make enough probe for 2 3 hybridizations Incubate at 70 C for 10 min Chill on ice Add the following reagents to the tube Incubate at 37 C for 2 hrs B Hydrolysis of RNA Mix 10pl 1N NaOH and 10pl 0 5M EDTA Add to RT reaction Incubate for 15 min at 65 C Neutralize 25pl 1M Tris ph 7 4 or 25pl 1M HEPES pH 7 0 OpArray User Manual C Reaction Cleanup Place 450pl H O in Microcon 30 Add reaction mix Spin at 11 000 x g for 12 min Remove flow through Repeat wash 2 x with H O Invert microcon in a new tube and elute by spinning 11 000 x g for 20 sec The volume may range from Apl to 40pl Dry down in spin vac and store at 20 C D Coupling of Cy Dye Resuspend cDNA in 4 5pl of H O Resuspend aliquot of Cy dye in 4 5pl 0 1 M Car
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