Dot Blot 96
Contents
1. 96 wells plus 2 bores for the guide pins Order No 053 410 1 Tube adapter for the collecting tank Im Vacuum tube Order No 049 001 1 Certificate of guarantee 1 Instruction Manual Dot Blot 96 System 230 V Order No 053 400 Dot Blot 96 System 115 V Order No 053 490 Dot Blot 96 System 100 V Order No 053 491 e Transfer unit consisting of Collecting tank volume 500 ml Filter plate support with 96 wells and 2 guide pins Cover plate with 96 wells and buffer reservoir volume 150 ml 4 Screws e Silicone sealing sheet sealing mat for the filter plate 96 wells plus 2 bores for the guide pins Order No 053 410 e 1 Tube adapter for the collecting tank e 1 Vacuum pump with flow valve and manometer Order No 049 000 230V 50 Hz or 049 090 115V 60 Hz or 040 091 100V 50 60 Hz e 1m Vacuum tube Order No 049 001 e 1 Certificate of guarantee e 1 Instruction Manual Ordering Information Item Order No Dot Blot 96 System with vacuum pump 230 V 053 400 Dot Blot 96 System with vacuum pump see above 115 V 60 Hz 053 490 Dot Blot 96 System with vacuum pump see above 100 V 50 60 Hz 053 491 Dot Blot 96 without vacuum pump 053 401 Vacuum pump MP86 with manometer and adjustable vacuum gauge 049 000 max delivery 6 l min end vacuum 100 mbar 230 V dto 115 V 60 Hz 049 090 dto 100 V 50 60 Hz 049 091 Accessories Silicone sealing sheet sealing mat 053 410 Srews 2 pcs 014 013 Vacuum tube 1m 049 001 Whatman2. filter paper 25 pieces 29 7 cm x 21 cm thickness 0 9mm 092 002 Whatman DEAE paper DE81 25 pieces 46 cm x 57 cm 3658915 Whatman Nitrocellulose WCN 15 pieces 16 cm x 16 cm pore size 0 45 um 090 001 Whatman nylon membrane 15 pieces 16 cm x 16 cm pore size 0 45 um 091 001 2 0 Safety and Operating Information Caution Don t use organic solvents or solvents with chaotropic ions for cleaning Solvent resistance Use neither organic solvents e g acetone chloroform nor gt 30 ethanol Don t use chaotropic ions e g urea guanidine hydrochloride Cleaning A detergent solution or 0 1 N NaOH solution or A dilute alcohol solution lt 30 Vacuum Maximum 500 mbar less than atmospheric pressure 3 0 Set Up 3 1 Unpack and Check Unpack and carefully examine the electrophoresis unit Report any damage to BIOMETRA Do not attempt to operate this device if physical damage is present Save all packing material if damage is found VAN Do not attempt to operate this device if physical damage is present N Attention A Please fill out and send back the warranty registration card This is important for you to claim to full warranty both parts and repair are covered within the full warranty period Place the chamber in proximity to the vacuum pump with which it is to be connected Be sure to place the chamber in a safe dry location away from the edge of the working surface 5 0 Assembling the Transfe
3. with deionised water Pay attention to the guide lines for cleaning given in chapter 2 0 Safety and Operating Informations 7 0 Application Information 7 1 Membrane Properties Nitro cellulose DNA RNA Proteins Hydrophobic Bond _ Protein Blot E A a Nylon DNA RNA Protein Hydrophobic Bond DNA RNA Blot eee ee el DEAE Cellulose Preparative Protein Blot Protein Sequencing DPT Cellulose Covalent Bond DEAE Diethylaminoethyl PVDF Polyvinylidendifluoride DBM Diazobenzyloxymethyl DPT Diazophenylthioether 7 2 Immunoblot Application Note Do not use gelatine for blocking as it clogs the membrane completely BSA or ovalbumine among others are suitable reagents Note Pay attention to the maximal binding capacity of your membrane during the trial application as otherwise the membrane can become clogged If the composition and concentration of your sample is unknown first conduct a dilution series with the Dot Blot 96 Note Do not ever let the membrane dry out completely during the individual incubation or washing steps Note When using the included vacuum pump aerate the Dot Blot 96 while the pump is running by loosening the knurled wheel on the manometer Reapply vacuum to the Dot Blot 96 apparatus by tightening the knurled wheel 1 When using nitro cellulose filters equilibrate the membrane for 5 10 minutes in the buffer solution which you use to wash your samples 2 After assemblin
4. Biometra a Whatman company Dot Blot 96 System Order No 053 400 490 491 Dot Blot 96 Order No 053 401 Manual January 1998 Warning VAN Please read these instructions carefully A N before using this apparatus Biometra biomedizinische Analytik GmbH Rudolf Wissell Stra e 30 D 37079 G ttingen P O Box 1544 D 37005 G ttingen Tel 49 0 5 51 50 68 6 0 Fax 49 0 5 51 50 68 6 66 e mail info biometra co uk internet http www biometra de 1 0 Intended Uses And Specifications The Dot Blot 96 concept is based on knowledge of scientists current requirements Therefore the device is appropriate for exceedingly different experimental approaches For the use of the Dot Blot 96 it is irrelevant whether the user desires to analyse proteins or nucleic acids and whether the sample determination is performed using antigen antibody complexes or via hybridisation with radioactive or non radioactive probes This multiplicity of applications makes the Dot Blot 96 a very valuable device in all cases where a standardised and reproducible assay of concentrated punctate samples is desired Typical examples for the application of the Dot Blot 96 are the detection of genomic DNA screening of bacterial DNA for recombinants screening of expression plasmids using an antibody the immunological detection of a protein in column fractions etc A newly designed sealing system makes the use of conventional sealing rin
5. e beyond the edges of the membrane otherwise the membrane will crack VAN Note Eliminate all air bubbles between the membrane filter paper and sealing mat by rolling a glass pipette or a similar object across the membrane 6 Place the cover plate with the buffer reservoir on the membrane so that it corresponds to the orientation of the guide pins 7 First tighten one pair of opposite screws and then the other pair Note Hand tighten the screws Do not use any tools 8 Apply vacuum to the transfer unit lt 150 mbar less than atmospheric pressure and tighten the screws again if necessary Check your vacuum by pipetting buffer solution into some or all of the wells and filtering it through the membrane Cover plate with 96 wells and buffer reservoir Screw Membrane Whatman 3MM filter paper Silicone sealing sheet Support es U Vacuum connector Stabilizer Fig 1 Set up of a Dot Blot 96 sandwich 6 0 Disassembling the unit 1 First interrupt the vacuum before opening the screws When using the vacuum pump delivered with the system Order No 049 000 090 091 you can apply ambient pressure by opening the flow valve 2 Remove the transfer buffer from the upper buffer reservoir Afterwards the apparatus can be disassembled in reverse order to assembling 3 Please rinse all parts of the Dot Blot 96 after use
6. ee from defects in materials or workmanship under normal use or service for 12 months from date of shipment If during this time this unit proves defective in materials or workmanship Biometra GmbH will repair or replace it free of charge if returned to us prepaid This guarantee does not cover damage in transit damage caused by carelessness misuse or neglect or unsatisfactory performance as a result of conditions beyond our control or consequential losses as a result of failure of our product Biometra biomedizinische Analytik GmbH Rudolf Wissell Strage 30 D 37079 Gottingen P O Box 1544 D 37005 G ttingen Tel 49 0 5 51 50 68 6 0 Fax 49 0 5 51 50 68 6 66 e mail info biometra co uk internet http www biometra de
7. g the apparatus see 5 rinse the membrane with buffer Rinsing solution Vacuum on 3 Pipette your samples into the individual wells Add samples Vacuum off Filter the samples through the membrane with a weak vacuum for Filtration approximately 30 minutes If the sample volumes are greater than 350 ul Vacuum on filtering time will take longer Allow the liquid which remains in the wells to be drawn off by the vacuum 4 Wash the membrane at least twice with wash solution 5 Pipette your blocking solution into the wells Filter the blocking solution through the membrane for about 30 minutes with a weak vacuum Allow the liquid which remains in the wells to be drawn off by the vacuum 6 Wash the membrane at least twice with wash solution 7 Pipette the primary specific antibodies Ab into the wells Filter the antibody solution through the membrane for about 30 minutes with a weak vacuum 8 Wash the membrane at least twice with wash solution 9 Pipette the secondary antibodies Ab into the wells Filter the antibody solution through the membrane for about 30 minutes through the membrane with a weak vacuum 10 Wash the membrane at least twice with wash solution 11 Remove the membrane from the Dot Blot 96 apparatus to perform further reaction steps for determining the samples Use this chart for the following blot determinations Glycoprotein Lectin Interaction Receptor Ligand Interaction Washing Vacuum
8. gs unnecessary and thus greatly facilitates the set up of the transfer unit The Dot Blot 96 can be assembled extremely quickly and is then ready for the application of samples Multiple channel pipettes can be used to apply valuable reagents e g antibodies nucleic acid probes as the arrangement of the 96 wells which are marked with A H and 1 12 corresponds to the format of microtiter plates These precisely milled wells volume 350 ul allow a reproducible application of the same sample volumes Washing sequences can be performed within minutes comfortably and without tedious pipetting To achieve this the wash solution is placed in a buffer reservoir which has a volume of 150 ml on the cover plate and is drawn through all 96 wells on applying vacuum The liquid which has passed through the membrane accumulates in the collecting tank and can be used for further analyses monitoring for radioactivity monitoring for antigens among others The generous dimensions of this tank give the device the required stability and normally permit the performance of several dots without having to empty the collecting tank Systems and Accessories Available Dot Blot 96 Order No 053 401 e Transfer unit consisting of Collecting tank volume 500 ml Filter plate support with 96 wells and 2 guide pins Cover plate with 96 wells and buffer reservoir volume 150 ml 4 Screws e 1 Silicone sealing sheet sealing mat for the filter plate
9. hern protocols Should you have any problems with this unit please contact our service department or your local Biometra dealer Biometra biomedizinische Analytik GmbH Service Department Rudolf Wissell StraBe 14 16 D 37079 G ttingen Phone Fax A 49 0 5 51 50 88 1 10 49 0 5 51 50 88 1 11 If you would like to send the unit back to us please read the following return instructions Instructions for return shipment e Return only defective devices For technical problems which are not definitively recognisable as device faults please contact the Technical Service Department at Biometra Tel 49 551 5068640 e Use the original box or a similarly sturdy one e Label the outside of the box with CAUTION SENSITIVE INSTRUMENT e Please enclose a precise description of the fault which also reveals during which procedures the fault occurred if possible e Important Clean all parts of the instrument from residues and of biologically dangerous A chemical and radioactive contaminants Please include a written confirmation use the Equipment Decontamination Declaration following on the next page that the device is free of biologically dangerous and radioactive contaminants in each shipment If the device is contaminated it is possible that Biometra will be forced to refuse to accept the device e The sender of the repair order will be held liable for possible losses resul
10. on Blocking Vacuum off Filtration Vacuum on Washing Vacuum on Add 1st Ab Vacuum off Filtration Vacuum on Washing Vacuum on Add 2nd Ab Vacuum off Filtration Vacuum on Washing Vacuum on 7 3 DNA and RNA Dot Blot Note Nitro cellulose and nylon bind nucleic acids at different salt concentrations Select application and wash solutions which are appropriate for the type of membrane you employ 1 Equilibrate the membrane for approximately 30 minutes in your application solution before placing it into the Dot Blot 96 apparatus 2 After the transfer unit has been assembled see Chapter 5 rinse the wells Rinsing twice with wash solution Allow a small amount of wash solution to remain in Vacuum on the wells 3 Pipette the prepared samples which have been diluted with application Add samples solution into the wells Vacuum off Filter the samples for approximately 30 minutes through the membrane using Filtration a weak vacuum Vacuum on 4 Wash the membrane at least twice with wash solution Washing Vacuum on 5 After the wash solution has flowed through the membrane maintain the Drying vacuum for an additional 5 minutes to dry the membrane Vacuum on 6 Remove the membrane from the Dot Blot 96 apparatus and allow it to dry completely at room temperature Further treatment of the membrane fixation prehybridisation hybridisation is identical to conventional Northern or Sout
11. r Unit AN Prior to use ensure that the 96 wells in the filter plate support are not clogged Before inserting a membrane into the Dot Blot 96 please read the information about pretreatment of the desired filter material given by the manufacturer 1 Screw the tube adapter into the housing of the collecting tank The vacuum pump can either be connected directly to the Dot Blot apparatus or indirectly via an intermediate wash bottle The use of a wash bottle is advisable when working with radioactive substances or very concentrated saline solutions e g 10 x SSC 2 Place the filter plate support stabilizer into the collecting tank 3 Align the silicone sealing sheet rubber mat with the guide pins on the filter plate support The 96 bores of the sealing mat must exactly correspond with the 96 wells in the filter plate support AN Note Wells which are not in use can be covered with appropriately cut strips of Parafilm which are laid onto the desired section of the sealing mat 4 Place a piece of Whatman 3MM filter paper which has been cut to size and equilibrated in a buffer solution on the sealing mat Use the wash solution which is employed for washing during the blot procedure as the buffer solution 5 The membrane which has been pre cut to fit your sample needs and equilibrated in buffer solution is laid on the filter paper Select the size of the membrane so that the guide pins on the filter plate support li
12. ting from insufficient decontamination of the device e Please enclose a note which contains the following 11 a Sender s name and address b Name ofa contact person for further inquiries with telephone number Equipment Decontamination Certificate To enable us to comply with german law i e 28 StriSchV 17 GefStoffV and 19 ChemG and to avoid exposure to hazardous materials during handling or repair will you please complete this form prior to the equipment leaving your laboratory COMPANY INSTITUTE ADDRESS TEL NO FAX NO E MAIL EQUIPMENT Model Serial No If on loan evaluation Start Date Finish Date Hazardous materials used with this equipment Has the equipment been cleaned and decontaminated YES NO delete Method of cleaning decontamination NAME POSITION HEAD OF DIV DEP INSTITUTE COMPANY SIGNED DATE PLEASE RETURN THIS FORM TO BIOMETRA GMBH OR YOUR LOCAL BIOMETRA DISTRIBUTOR TOGETHER WITH THE EQUIPMENT PLEASE ATTACH THIS CERTIFICATE OUTSIDE THE PACKAGING INSTRUMENTS WITHOUT THIS CERTIFICATE ATTACHED WILL BE RETURNED TO SENDER 14 Warranty This laboratory instrument is produced with the highest practical standards of materials workmanship and design The design and manufacture of parts have been conceived with one purpose to produce units which will give satisfactory service Biometra GmbH guarantees this unit to be fr
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