Investigation Title Transcription profiling of E. coli HMS174(DE3)(pET30aNproGFPmut3.1) to analyze cellular response to limited induction with IPTG Comment[Submitted Name] Transcription profiling of E. coli HMS174(DE3)(pET30aNproGFPmut3.1) to analyze cellular response to limited induction with IPTG Experimental Design time_series_design transcription profiling by array Experimental Design Term Source REF The MGED Ontology EFO Comment[AEMIAMESCORE] 5 Comment[ArrayExpressReleaseDate] 2010-03-26 Comment[ArrayExpressAccession] E-MARS-17 Comment[MAGETAB TimeStamp_Version] 2011-06-28 14:25:18 Last Changed Rev: 14857 Experimental Factor Name time Experimental Factor Type time Experimental Factor Term Source REF Person Last Name Striedner Scharl Rader Person First Name Gerald Theresa Robert Person Mid Initials Person Email gerald.striedner@boku.ac.at theresa.scharl@boku.ac.at robert.rader@tugraz.at Person Phone not available not available not available Person Fax Person Address Person Affiliation BOKU - Institute of Applied Microbiology BOKU - Institute of Applied Microbiology Institute for Genomics and Bioinformatics Person Roles submitter data_analyst data_coder Person Roles Term Source REF The MGED Ontology mo Quality Control Type dye_swap Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2010-03-26 PubMed ID Publication DOI Publication Author List Publication Title Publication Status Publication Status Term Source REF Experiment Description This transcription profiling time course experiment was conducted in order to analyze the cellular response of the T7 RNA polymerase based E. coli expression system HMS174(DE3)(pET30aNproGFP) to a limited induction with isopropyl-beta-D-galactoside (IPTG). The used cell material was produced under well controlled and defined conditions in an exponential carbon limited fed-batch cultivation at a growth rate of 0.1 per hour. One doubling past feed start induction was started by continuously feeding limiting amounts of inducer gaining in a constant IPTG to cell dry weight ratio of 1µmol/g. Due to the low induction level a physiologically tolerable recombinant gene expression rate was generated and production period was significantly elongated. Non induced cells sampled at the time point of induction were used as reference and compared to cells subsequently sampled during the production period (sampling frequency 2 hours). Protocol Name LSID:MAGE:genome.tugraz.at:mars:at.tugraz.genome.marsejb.utils.vo.ProtocolVO:985 LSID:MAGE:genome.tugraz.at:mars:at.tugraz.genome.marsejb.utils.vo.ProtocolVO:1130 LSID:MAGE:genome.tugraz.at:mars:at.tugraz.genome.marsejb.utils.vo.ProtocolVO:1131 LSID:MAGE:genome.tugraz.at:mars:at.tugraz.genome.marsejb.utils.vo.ProtocolVO:984 LSID:MAGE:genome.tugraz.at:mars:at.tugraz.genome.marsejb.utils.vo.ProtocolVO:1125 LSID:MAGE:genome.tugraz.at:mars:at.tugraz.genome.marsejb.utils.vo.ProtocolVO:1144 Protocol Type specified_biomaterial_action grow nucleic_acid_extraction labeling hybridization lowess_group_normalization Protocol Description Title: RNA sample drawing. Description: CHEMICALS AND EQUIPMENTPhenol (Tris-HCl-saturated, pH= 6.7 ? 0.2) [Sigma P-4557]EtOH absolute ý [Sigma; #32221] M: 46.07 g/lSyringes sterile (10 ml)RNase-free reaction tubes 1.5 mlEppendorf Centrifuge 5415RREAGENTS TO PREPAREPhenol/Ethanol stabilising solution (5%)Mix 2.5 ml of phenol with 47.5 ml of EtOH absolute (calculate required volume of solution for each cultivation and modify if necessary (2ml per sample taking)).PROCEDUREThis protocol can be used for RNA-sample drawing in E. coli cultivations. Approximately 3 mg of biomass dry matter will be drawn of the bioreactor and immediately stabilised with the solution prepared above. Always use RNase-free tips and reaction tubes, wear nitril-gloves. All sample preparation steps should be carried out under the extractor hood.- Pipette 2 ml of phenol/ethanol stabilising solution into 10 ml syringe- Pull plunger to a point on the scale- Draw accurately 4 ml of fermentation broth into the syringe and shake immediately - Using the formula (1) calculate volume of suspension for aliquoting into cooled 1.5 ml RNase-free reaction tubes (use the theoretical biomass dry matter of the fermentation and prepare before; tubes should contain 3 mg of bdm) - Centrifuge samples at 13400 g and 4ýC for 2 min- Discard supernatant- Store at -80ýC. (1)REVISIONVersiongenerated onfrommodifications123.03.2004Striednern.A.226.05.2008MarischTranslation to englishAUSTRIAN CENTER OF BIOPHARMACEUTICAL TECHNOLOGYAuthor:StriednerHelgaDate:23.03.200422.12.Working groupMO-FermentationPROTOCOL: SAMPLE TAKING PROCEDURE OF RNA IN E. COLI CULTIVATIONSVersion:2PAGE 1 OF 1ý MO-Ferm AG Title: Cultivation conditions PS19. Description: DURCHFýHRUNGSDATUM : Start Batch 14.7.2004 8:30 End of process 15.7.2004 12:30 REACTOR: 20 l MBR Bioreactor (14l working volume)PROCESS CONTROL SPS: Siemens Simatic S7 (Siemens Automation)SCADA: customized process automation designed by ISE (Industrial Software Engineering GmbH, Vienna) based on Proficy HMI SCADA - iFIX 4.0 (GE Fanuc Intelligent Platforms Europe S.A., Darmstadt Germany)process data storage: Proficy iHistorian 3.0 ON-LINE MONITORING DEVICES AND CONTROL STRATEGIES Manipulated variables pH probe: Mettler Toledo, pH2100e, InFit 761e control: pH control by addition of 25 % ammonia solution (MERCK) setpoint: 7 ý 0.05 pO2 probe: Mettler Toledo InPro?6800 control: control of by stirrer speed and aeration rate setpoint: dissolved oxygen level > 30% Temperature probe: Pt100 (Thermoest, Vienna, A) control: double jacket cooling/heating system setpoint: 37 ýC ý 0.05 Measured variables CO2 and O2 offgas measurement Advanced Optima gas analyzer, Hartmann and Braun, ABB Austria MEDIUMMinimal medium: 3 g KH2PO4 and 6 g K2HPO4.3H2O per liter (buffer capacity and also P and K sources). All other components added in relation to the gram cell dry weight to be produced: 0.25 g sodium citrate (trisodium salt.2H2O; ACROS organics), 0.10 g MgSO4.7H2O, 0.02 g CaCl2.2H2O, 50 ýl trace element solution, and 3 g glucose.H2O.The trace element solution was prepared in 5 N HCl and contained (g/l) 40.0 FeSO4.7H2O, 10.0 MnSO4.H2O, 10.0 AlCl3.6H2O, 4.0 CoCl2 (Fluka), 2.0 ZnSO4.7H2O, 2.0 Na2MoO2.2H2O, 1.0 CuCl2.2H2O, and 0.50 H3BO3.INDUCTION Strategy inducer feed (exponential profile) Induction level limited induction 1ýmol/g cell dry weight Time point of induction one doubling past feed start CULTIVATION MODEBatchThe minimal medium for batch processes is supplemented with 0.15 g yeast extract per gram CDW to be produced to accelerate initial growth of the population. For inoculation, 1 ml of a working cell-bank is transferred aseptically to the bioreactor. Foaming was suppressed by addition of 0.5 ml antifoam suspension (Glanapon 2000, Bussetti, Vienna) per litre medium. Settings: Batchvolume: 4 l Cell dry weight: 30 g Exponential FeedThe minimal medium was prepared according to the amount of cell dry weight to be produced during the feeding phase. Feeding was initiated when the culture entered the stationary phase. A fed-batch regime with an exponential substrate feed was used to provide a constant growth rate of 0.1h-1. The substrate feed was controlled by increasing the pump speed according to the exponential growth algorithm, x = x0.eýt, with superimposed feedback control of weight loss in the substrate tank. Foaming was suppressed by addition of 0.5 ml antifoam suspension (Glanapon 2000, Bussetti, Vienna) per litre feed medium. Settings Fedvolume: 8 l Doublings: 4 Growth rate: 0.1 h-1 Cell dry weight total: 480 g AUSTRIAN CENTER OF BIOPHARMACEUTICAL TECHNOLOGYAuthor:StriednerHelgaDate:22.12.Working groupMO-FermentationCULTIVATION PS19Version:1PAGE 1 OF 2ý MO-Ferm AG Title: RNA Extraction - DNA digestion. Description: PROTOCOL: RNA EXTRACTION AND DNA DIGESTION GESAMTER RNA FROM E. COLIEQUIPMENT AND CHEMICALSNuclease-free water [Ambion, #9932]EDTA (ethylene diamine tetraacetic acid) [Merck, #108418] M: 372.24 g/lNaOH pellets, extra pure [Merck; #106482]Lysozyme [Amresco, #0663-5G] Trizol [Invitrogen, #15596-018]Chloroform [Sigma-Aldrich; #C-2432]Isopropanol [Sigma-Aldrich; #I-9516]Tris [Roth; #5429.1] M: 121.14 g/lEtOH absolute ý [Sigma; #32221] M: 46.07 g/lHydrochloric acid (32 %) [Merck, #100319]DNaseI with 10x buffer [Roche, #04716728001]Phenol-chloroform-isoamylalcohol [Invitrogen, #15593-031]NaCl [Merck, #106404] M: 58.44 g/lEtOH absolut ý [Sigma; 32221] M: 46.07 g/lEppendorf Centrifuge 5415RScientific Industries Vortex Genie 2NanoDrop ND1000Eppendorf Thermomixer comfortREAGENTS TO PREPAREAll solutions should be prepared with nuclease-free water. Glass ware should be baked at 180ýC over night. Always use gloves and RNase-free tips. Extraction steps must be carried out under the extractor hood.0.5 M EDTA (pH= 8.0)Dissolve 18.6 g of EDTA in 80 ml nuclease-free H2O, adjust the pH to 8.0 with NaOH pellets and fill up to 100 ml with nuclease-free water.1M Tris-HCl (pH= 7.4)Dissolve 60.57 g of Tris in 400 ml nuclease-free H2O, adjust the pH with 37% HCl to 7.4 and fill up to 500ml with nuclease-free water.TE-buffer (pH= 7.5)Mix 1 ml of 0.5M EDTA (pH= 8.0) and 5 ml 1M Tris-HCl (pH= 7.4) and fill up to 500 ml with nuclease-free water.Lysozyme solution (10 mg/ml)Dissolve 100 mg of lysozyme in 10 ml of TE-buffer. Aliquote per 500 ýl in RNase-free reaction tubes.Ethanol (75 %)Mix 37.5 ml of EtOH absolute with 12.5 ml of nuclease-free water.PROCEDUREThis protocol can be used for samples taken during fermentations according to the sample taking protocol for RNA samples. Samples should contain 3 mg/mg BDM per aliquot. Samples should be stored at -80ýC. The RNA-extraction procedure is adjusted to a 1.5 ml scale. - Unfreeze and vortex pellet (1 min on the vortex)- Add 100ýl of lysozyme solution (10mg/ml in TE-buffer) - Vortex pellet for 5 min at room temperature to resuspend cell pellet- Add 800 ýl of trizol and vortex- Incubate for 5 min at room temperature without vortexing- Add 160ýl of chloroform and vortex for 1 min at room temperature- Incubate for 2 min 30 sec at room temperature- Centrifuge at 12.000 g (13400 rpm) for 15 min at room temperature- Transfer the upper aqueous phase (500 ýl) to a new RNase free reaction tube - Add 2 volumes of isopropanol (1000 ýl)- Incubate for 15 min at room temperature- Centrifuge at 12.000 g (13400 rpm) for 15 min at 4ýC- Discard supernatant and wash pellet with 1 ml of 75% EtOH. (RNA can be stored at this step at -20ýC until use.)- Centrifuge at 12.000 g (13400 rpm) for 15 min at 4 ýC- Discard supernatant- Air-dry pellet (or at 37ýC on a hot-block) and dissolve in 300 ýl of nuclease-free waterConcentration of isolated RNA is measured with the NanoDrop and the quality should be controlled with the Bioanalyzer according to the manufacturerýs protocols. PROTOCOL: DNA-DIGESTION OF E. COLI RNA SAMPLESEQUIPMENT AND CHEMICALSNuclease-free water [Ambion, #9932]DNaseI with 10x buffer [Roche, #04716728001]Phenol-chloroform-isoamylalcohol [Invitrogen, #15593-031]Chloroform [Sigma-Aldrich; #C-2432]NaCl [Merck, #106404] M: 58.44 g/lEtOH absolut ý [Sigma; 32221] M: 46.07 g/lEppendorf Centrifuge 5415RScientific Industries Vortex Genie 2NanoDrop ND1000Eppendorf Thermomixer comfortREAGENTS TO PREPAREAll solutions should be prepared with nuclease-free water. Glass ware should be baked at 180ýC over night. Always wear gloves and RNase-free tips for working with RNA.3 M NaCl Dissolve 1.75 g of NaCl in 10 ml of nuclease-free water and aliquot per 500 ýl.Ethanol (70 %)Mix 35 ml of EtOH absolute with 15 ml of nuclease-free water.PROCEDUREThis protocol can be used for RNA samples prepared with RNA isolation protocol. Concentration of RNA should be measured with the NanoDrop according to the manufacturerýs protocol. Samples should be stored at -80ýC. The DNA-digestion procedure is adjusted to a 1.5 ml scale. DNA digestion mix:Prepare a reaction mix containing:- RNA (400 ýg) 250ýl- 25 mM MgCl2 60ýl- 200mM Tris Buffer 33 ýl- DNaseI (10 U/ýl) 7 ý- Incubate at 30ýC for 30 min on a hot-block- Add nuclease-free water to RNA digestion mix to obtain a final volume of 350 ýl- Add 350 ýl of phenol-chloroform-isoamylalkohol - Vortex 1 min and centrifuge 2 min at 13400 rpm- Transfer 300 ýl of upper aqueous phase to a new reaction tube- Add 300ýl of chloroform - Vortex 1 min and centrifuge 2 min at 13400 rpm- Transfer 250 ýl of upper aqueous phase to a new reaction tube- Add 25 ýl of 3M NaCl and 500 ýl of EtOH absolute and mix- Incubate for 45 min or over night at -20ýC- Centrifuge at 13400 rpm for 10 min at 4ýC- Discard supernatant and wash pellet with 500 ýl of 70% EtOH. - Centrifuge at 13400 rpm for 10 min at 4 ýC- Discard supernatant- Air-dry pellet (or at 37ýC on a hot-block) and dissolve in 34 ýl of nuclease-free waterConcentration of isolated and digested RNA is measured with the NanoDrop and the quality of RNA should be controlled with the Bioanalyzer according to the manufacturerýs protocols. Title: Reverse Transcription and Indirect Labeling. Description: PROTOCOL: REVERSE TRANSCRIPTION AND INDIRECT LABELLING OF TOTAL RNA FOR cDNA AND OLIGO ARRAYS=============================================================================================COMMENTS=========The samples to be compared are each labelled with a different fluorescent dye and then subjected to paired competitive hybridisations. Indirect labelling involves two steps, firstly, incorporation of amino-allyl dUTP by reverse transcription, and then attachment of the fluorescent dyes.The following is an adaptation of a protocol from the Ajioka group within the Department of Pathology, University of Cambridge. This protocol can be used to label as little as 3 ýg of total RNA. However, the following is for up to 30 ýg of total RNA. For each microarray slide you should have two target samples; one labeled with Cy3, the other with Cy5. Normally 10 ýg of RNA should be used as a starting material.Microcon YM-30 column steps are approximate, and need to be optimized for your particular centrifuge. If you centrifuge for too long and the pellet is dry, reload waste and re-centrifuge.EQUIPMENT AND REAGENTS====================== - dATP, dCTP, dTTP and dGTP (Sigma; Cat. No. dNTP-100A) - Amino-allyl dUTP (Sigma; Cat. No. A0410) - Oligohexamere - Nuclease free water - Cy3 and Cy5 mono-reactive dye pack, (Amersham; Cat. No. PA23001 and PA25001) - RNAsin (Promega; Cat. No. 18064-014) - Superscript III Reverse Transcriptase (Invitrogen; Cat. No.18080-044) - EDTA (BDH; Cat. No. 100935V) - Sodium hydroxide, (BDH; Cat. No. 102525P) - Tris-HCl solid (BDH; Cat. No. 443864E) - Sodium Carbonate buffer 0.2M (pH 8,5-9,0) - Microcon YM-30 concentrators (Millipore; Cat. No. 42410) - MinElute PCR Purification Kit (Qiagen; Cat. No. 28004) - Hydroxylamine (Sigma; Cat. No. H-2391) - DMSO, high purity - Sonicated Salmon Sperm DNA (Invitrogen; Cat. No. 15632-011) - Centrifuge - Hot-block - Speed VacREMOVAL OF RNASE================All materials should be autoclaved and only handled using gloves to avoid RNase contamination. Glassware should be baked at 180 ýC overnight. MilliQwater and solutions should be nuclease free. The work area can be cleaned using RNase Zap to further limit the risk of RNase contamination. If possible, keep a set of pipettes purely for RNA work.PROCEDURE =========Preparation of 0.2 M Sodium Carbonate Buffer: pH 8.5 - 9.0. * Solution I: Dissolve 0.84g NaHCO3 in 50 mL DEPC H2O in a disposable sterile Falcon tube. * Solution II: Dissolve 1.05g Na2CO3, in 50 mL DEPC H2O in a disposable sterile Falcon tube. * Mix 45 mL of Solution I and 2.75 of mL Solution II in a disposable sterile Falcon tube. * Check the pH by aliquoting 5 mL into a new 15 mL Falcon tube, if needed adjust the pH by adding appropriate amount of Sol-I or Sol-II. Never check the pH directly in the stock solution as that is a common source of RNAase contamination. * Aliquot 0.2 mL into RNAase-free tubes, store at -20ýC. Discard the tube after use or 24 hours, whichever comes first. Reagents to mix and aliquot: 1. Make a 50 x dNTP mix - 10 ýl dATP (100 mM stock) - 10 ýl dCTP (100 mM stock) - 10 ýl dGTP (100 mM stock) - 4 ýl dTTP (100 mM stock) - 6 ýl amino-allyl dUTP (100 mM)Then mix together and add to the master mix as below. 2. Master Mix for one sample (total 14.6 ýl) - 6 ýl First strand buffer (comes with Superscript III) - 0.6 ýl 50x dNTP mix made above - 3 ýl 0.1 M DTT (provided with Superscript III) - 0.25 ýl RNAsin - 2.75 ýl RNase-free water - 2 ýl Superscript IIIYou can make a pre-mix without Superscript III and make aliquots before storing at -20 ýC. Add Superscript III before use.Reverse transcription for amino-allyl dUTP incorporation: 3. RNA is isolated and purified using our standard protocol 4. Adjust the volume of RNA (normally 10 ýg) to 12.5 ýl with RNase-free water and add to a 1.5 ml RNase/DNase free microfuge tube 5. Add 3 ýl of anchored Oligo hexamer primer to each RNA sample 6. Place the tubes at 65 ýC for 10 minutes 7. Place the tubes immediately on ice for 5 minutes 8. Add 14.6 ýl of the master mix to each tube (made above) and incubate at 42 ýC for 2hrs 9. Remove the tubes from hot block, and add 10 ýl 0.5 M EDTA and 10 ýl of 1 M soduim hydroxide. Place at 65 ýC for 15 minutes 10. Remove from heat and place at room temperature for 2 minutes 11. Add 25 ýl of 1 M Tris-HCl (pH 7.5), mix 12. Add 450 ýl of RNase-free water to each sample and add each sample to a microcon-YM30 concentrator. Add sample without touching the membrane. 13. Centrifuge at room temperature at 13,000 rpm for about 7 minutes (or until about 50 ýl of water left in reservoir), empty waste, and then add another 400 ýl of water. 14. Centrifuge again at room temperature at 13,000 rpm for 7 minutes 15. Empty waste and add 400 ýl water for a third time 16. Centrifuge tube at room temperature at 13,000 rpm for 8 minutes (until volume left reached about 10 ýl). 17. Invert the column into new 1.5 ml collection tube. 18. Centrifuge at RT at 3,000 rpm for 4 minutes. 19. Place the tubes in the speed vac to dry on medium heat. Can be stored at -20 ýC.Dye attachment: 20. Resuspend the pellet in 4.5 ýl 0.2 M Sodium Carbonate buffer (pH 8.5-9.0). 21. If necessary resuspend one aliquot of Cy3 or Cy5 dye in 36.5 ýl of DMSO (sufficient for 8 labelling reactions), vortex and spin content down 22. Mix 4.5 ýl of resuspended dye with resuspended pellet. 23. Place in the dark for 1 hour at 23 ýC (in a hot block).Dye quenching and removal of unincorporated dye 24. Add 4.5 ýl of 4 M hydroxylamine to each sample to stop reaction and incubate in the dark for 15 minutes at 23 ýC.Clean up samples using Qiagen MinElute PCR Purification Kit: 25. Add 35 ýl of 3 M sodium acetate (pH 5.2) to each reaction, and mix both samples (Cy3 and Cy5) in a new 1.5 ml microfuge tube 26. Add 5 volumes (500 ýl) of buffer PB and mix. 27. Place a MinElute column in a provided 2 ml collection tube. 28. To bind DNA, apply the sample to the MinElute column and centrifuge for 1 minute at 13,000 rpm. 29. Discard flow through, and add 750 ýl of Buffer PE to the column. 30. Centrifuge for 1 minute at 13,000 rpm. 31. Repeat steps 29 and 30. 32. Discard flow through and place MinElute column back in same tube and centrifuge empty column for 1 minute at 13,000 rpm. 33. Place column into a new 1.5 ml microfuge tube and add 10 ýl elution buffer (nuclease free water). 34. Incubate at room temperature for 1 minute in dark. 35. Centrifuge for 1 minute at 13,000 rpm. 36. Add another 10 ýl of RNase-free water to column and centrifuge for 1min at 13,000rpm. Collect in same tube. 37. Dry in the speed vac to a volume of 2 to 5 ýl (about 10 minutes). 38. Add 2 ýl sonicated salmon sperm DNA (from 10 mg / ml stock).The two samples (i.e. sample and control) have been combined together for hybridisation to a microarray and the blocking agent, sonicated salmon sperm DNA, has been added. This material should now be used immediately to prevent any decay. Please refer to the appropriate hybridisation protocol for the next steps.PROTOCOL VERSION================1.0 / 14.12.2007Author: Marisch, KarolineAUSTRIAN CENTER OF BIOPHARMACEUTICAL TECHNOLOGY Title: Hybridization MWG arrays. Description: PROTOCOL: HYBRIDIZATION OF MICRO-ARRAYS ON TECAN HS400 STATIONEQUIPMENT AND CHEMICALSAlbumin-Fraction V (BSA) [Applichem, A1391] M ~ 68000g/molSDS 10% (Dodecylsulfate sodiumsalt) [Merck , #106022]SSC Buffer (20x) [Invitrogen, #15557-044]SSPE Buffer (20x) [Fluka, #85637]MWG Hybridisationbuffer [MWG # 1180-200000] Custom MWG Array [MWG E. coli K12 V2 oligo set, Spotting ARC Seibersdorf]HQ-H2O Eppendorf Thermomixer comfortTecan HS400REAGENTS TO PREPAREAll solutions are calculated for the use of all four slides in the TECAN HS400.Blocking buffer (400 ml): 4x SSC 80 ml 20x SSC0.5% (v/v) SDS 20 ml SDS 10%1% BSA 4g BSAHQ-H2O 300 mlFiltrate solution through a 0.22 ým filter.Pre-hybridization buffer:5x SSPE 50 ml 20x SSPEHQ-H2O 150 mlWashing buffer 1:2x SSC 50 ml 20x SSC0.1% SDS 5 ml SDS 10%HQ-H2O 445 ml Washing buffer 2:1x SSC 25 ml 20x SSCHQ-H2O 475 mlWashing buffer 3:0.5xSSC 12.5 ml 20x SSCHQ-H2O 487.5 mlPROCEDURESample preparation:Add MWG HybSolution to the already labelled and already mixed samples to obtain a final volume of 80 ýl (volume of the hybridization chamber). * Heat samples for 3 min at 95ýC on a heating-block.Preparation of Tecan HS400:Carefully read and follow all instructions shown on the screen and be very careful at sample injection steps. A ý+ý marks the chamber into which the sample should be injected and this has to be confirmed after each injection step on the station by pressing the OK button.* Open HS400 control application* Start a program* Connect the instrument* Put all tubes in the bottles. * Check numbers on tubes and bottles.* Prime the first used channel (5) to fill the tube* Switch on heating if necessary* Switch on the nitrogen supply* Open hybridization shell* Insert chambers into positions* Insert slides into slide positions, insert dummy slides into all unused positions* Close hybridization shell * Insert connectors* Start program and follow the instructions on the display carefully.* Select only chambers which are used during hybridization run.PROGRAM START1.WASH: Temp. ýC:42.0,First: Yes, CH.:5, Runs 1, Wash time: 0:00:30, Soak time: 0:00:302.HYBRIDIZATION: Temp. ýC:42.0, Agitation Frequency: High, Time: 0:07:003.WASH: Temp. ýC:42.0,First: No, CH.:5, Runs 1, Wash time: 0:00:30, Soak time: 0:00:304. HYBRIDIZATION: Temp. ýC:42.0, Agitation Frequency: High, Time: 0:07:005.WASH: Temp. ýC:42.0,First: No, CH.:5, Runs 1, Wash time: 0:00:30, Soak time: 0:00:306.HYBRIDIZATION: Temp. ýC:42.0, Agitation Frequency: High, Time: 0:07:007.WASH: Temp. ýC:42.0,First: No, CH.:5, Runs 1, Wash time: 0:00:30, Soak time: 0:00:308.HYBRIDIZATION: Temp. ýC:42.0, Agitation Frequency: High, Time: 0:07:009.WASH: Temp. ýC:42.0,First: No, CH.:5, Runs 1, Wash time: 0:00:30, Soak time: 0:00:3010.HYBRIDIZATION: Temp. ýC:42.0, Agitation Frequency: High, Time: 0:07:0011.WASH: Temp. ýC:42.0,First: No, CH.:6 Runs 6, Wash time: 0:03:00, Soak time: 0:01:0012.WASH: Temp. ýC:42.0,First: No, CH.:1 Runs 1, Wash time: 0:01:00, Soak time: 0:01:0015.PROBE INJECTION: Temp. ýC:50.0 -> 80ýl sample16.HYBRIDIZATION: Temp. ýC:50.0, Agitation Frequency: Medium, Time: 16:00:0017.WASH: Temp. ýC:42.0,First: No, CH.:2, Runs 4, Wash time: 0:001:00, Soak time: 0:03:0018.WASH: Temp. ýC:42.0,First: No, CH.:3 Runs 1, Wash time: 0:01:00, Soak time: 0:03:0019.WASH: Temp. ýC:30.0,First: No, CH.:3 Runs 4, Wash time: 0:01:00, Soak time: 0:03:0020.WASH: Temp. ýC:30.0,First: No, CH.:4 Runs 4, Wash time: 0:01:00, Soak time: 0:03:0021.SLIDE DRYING: Temp. ýC:30.0, Time: 0:02:00, Final Manifold Cleaning: No, Ch.: NoPROGRAM ENDLiquid identification:Liquid for channel 1: prehybridization bufferLiquid for channel 2: 2xSSC, 0.1% SDSLiquid for channel 3: 1xSSCLiquid for channel 4: 0.5xSSCLiquid for channel 5: blocking bufferLiquid for channel 6: HQ-H2OAfter Hybridization:After the slide hybridization a pop-up message appears (Run finished OK). Quit message and take out slides for scanning. * Insert dummy slides into all slide positions and close chamber shell* Start the ýsds-washý program to remove remaining contaminations and follow the instructions carefully PROGRAM START 1.WASH: Temp. ýC: 40.0, First: YES; CH.: 5; Runs: 5, Wash time: 0:01:00, Soak time: 0:02:00 2.WASH: Temp. ýC: 40.0, First: YES; CH.: 6; Runs: 5, Wash time: 0:02:00, Soak time: 0:02:00 3.SLIDE DRYING: Temp. ýC:40.0, Time: 0:00:30, Final Manifold Cleaning: No, Ch.: No PROGRAM END Liquid identification: Liquid for channel 1: Liquid for channel 2: Liquid for channel 3: Liquid for channel 4: Liquid for channel 5: 0.1% SDS Liquid for channel 6: HQ-H2O* After the ýsds-washý program is finished the RINSE step should be run.* All tubes should be inserted into HQ-water* After the display message: ýremove all tubes from liquidý this should be done and the final system drying step should be started* After finishing RINSE the chambers can be removed from the HS400.* Disconnect the instrument.* Switch of the heating if previously turned on.* Put chambers into HQ-water and incubate for about 1 h at 60ýC.* Dry chambers with nitrogen and store them until next use. Title: Normalization MWG. Description: ----------------------CARMAweb Normalization----------------------Background Subtraction Method: subtractWithin Slide Normalization Method: printtiploessBetween Slide Normalization Method: noneAverage Signal: meanReplicate Handling: yesUse Dye Swap Pairs: yesUse Dye Swap Filter: yesDye Swap Filter CutOff: 1Averaging Method: meanAverage Spots: no------------------------------------------------------------------------------------------------------------------------------------------------> sessionInfo()R version 2.2.1, 2005-12-20, x86_64-redhat-linux-gnuattached base packages:[1] tools methods stats graphics grDevices utils[7] datasets baseother attached packages:geneplotter annotate pgUtils RdbiPgSQL Rdbi maDB 1.8.0 1.8.0 1.2.0 1.4.0 1.4.0 1.7.4 affy Biobase limma 1.8.1 1.8.0 2.2.0 Protocol Parameters Protocol Hardware Protocol Software Protocol Contact Protocol Term Source REF SDRF File E-MARS-17.sdrf.txt Term Source Name The MGED Ontology ArrayExpress The MGED Ontology EFO mo Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version