ELITechGroup S.p.A. C.so Svizzera, 185 10149 Torino ITALY Offices: Tel. +39-011 976 191 Fax +39-011 936 76 11 E. mail: emd.support@elitechgroup.com WEB site: www.elitechgroup.com NOTICE of CHANGE dated 28/05/15 IMPORTANT COMMUNICATION FOR THE USERS OF PRODUCT: code In the Instruction for Use Manual (IFU), SCH m, some changes were introduced regarding the classification and labeling of substances / mixtures contained in the kit in accordance with Regulation n. 1272/2008 (CLP) and subsequent amendments. All changes are reported in detail in the enclosed Instructions for Use (IFU) and in the Material Safety Data Sheets related to the product. The assay analytical principle and test reagents are unchanged. PLEASE NOTE LA REVISIONE DI QUESTO IFU E COMPATIBILE ANCHE CON LA VERSIONE PRECEDENTE DEL KIT (vedi Package Insert incluso nel kit) THE REVIEW OF THIS IFU IS ALSO COMPATIBLE WITH THE PREVIOUS VERSION OF THE KIT (see Package Insert included in the kit) CET IFU MIS A JOUR ANNULE ET REMPLACE ET EST PARFAITEMENT COMPATIBLE AVEC LA VERSION PRECEDENTE DU KIT (voir Package Insert inclus dans le kit) LA REVISIÓN DE ESTE IFU ES COMPATIBLE TAMBIÉN CON LA VERSIÓN ANTERIOR DEL KIT (véa Package Insert incluido en el kit) A REVISÃO DO ESTE IFU ÉTAMBÉM COMPATÍVEL COM A VERSÃO ANTERIOR DO KIT (ver Package Insert incluído no kit) DIE REVIEW VON DIESER IFU IST KOMPATIBLE MIT DER VORIGE VERSION VON DEM TEST-KIT (Sehen Sie Package Insert im Kit enthalten) Instruments accessories and kits Specifics accessories and/or kits are intended to be used in association with instruments for automatic extraction and amplification as indicated in the User s manual, in Other products required section. Accessories and/or kits requirement must be expressly indicated in the purchase order of present amplification kit. Notice of Change nr. SCH m_04_en dated 28/05/15
ELITechGroup S.p.A. C.so Svizzera, 185 10149 Torino ITALY Offices: Tel. +39-011 976 19 Fax +39-011 936 76 11 E. mail: emd.support@elitechgroup.com WEB site: www.elitechgroup.com ASSAY PRINCIPLE The electrophoretic detection procedure involves preparation of amplification reaction products and loading them into agarose gel wells submerged in the electrophoresis buffer in the electrophoresis tank. When a direct current is applied to the electrophoresis tank, the DNA available in each well migrates towards the positive electrode and incorporates the ethidium bromide, a fluorophore present in agarose gel. The molecular lattice of the agarose gel allows the separation of smaller DNA molecules, i.e. with a lower number of base pairs (bp), which migrate more rapidly, from larger molecules, i.e. with a higher number of base pairs, which migrate more slowly. DNA may be viewed by exposing the gel to ultraviolet radiation and appears as an orange line in the shape of the well (band). The presence of the specific product of an amplification reaction means that target DNA is present in the starting sample. TABLE OF CONTENTS +2 C +8 C KIT DESCRIPTION The kit provides 8-well Ready Gel, in a tray and ready for use, electrophoresis buffer 50x to be reconstituted with sterile bidistilled water, loading buffer 6x for preparing the amplification reaction products, HinfI Marker as the molecular weight standard. The kit enables 64 detections, including molecular weight standards and controls. INTENDED USE page 1 ASSAY PRINCIPLE page 2 KIT DESCRIPTION page 2 MATERIALS PROVIDED IN THE KIT page 2 MATERIALS REQUIRED BUT NOT PROVIDED IN THE KIT page 3 OTHER PRODUCTS REQUIRED page 3 WARNINGS AND PRECAUTIONS page 3 SAMPLES AND CONTROLS page 4 PROCEDURE page 5 PROCEDURE LIMITATIONS page 10 PERFORMANCE CHARACTERISTICS page 10 REFERENCES page 11 TROUBLESHOOTING page 11 SYMBOLS page 12 INTENDED USE is a system for the detection and identification of DNA through electrophoretic separation on agarose gel of the products of nucleic acid amplification assays of the «OligoMIX Alert kit» series by ELITechGroup S.p.A. The kit is intended for use in detecting the results of qualitative amplification assays of nucleic acids for the detection of a target DNA in DNA extracted from biological samples or in cdna obtained from RNA extracted from biological samples. MATERIALS PROVIDED IN THE KIT Component Description Quantity Composition Labelling Gel Pronto 8 pozzetti 8-well Ready Gel Tampone di Elettroforesi 50x Electrophoresis buffer 50x Tampone di Caricamento 6x Loading buffer 6x Marker HinfI HinfI Marker Agarose gel with 8 wells in TAE buffer 1x with ethidium bromide TAE buffer 50x, to be diluted 50 times in water Loading buffer to be diluted 6 times in the sample Molecular weight standards, ready for use 8 Agarose, TRIS base, TRIS acetate, EDTA, 0.05% ethidium bromide 1 x 50 ml TRIS base, TRIS acetate, EDTA 1 x 0.5 ml 1 x 0.1 ml Glycerol, TRIS base, TRIS hydrochloride, EDTA, Bromophenol Blue, Xylencyanol FF Plasmid, Glycerol, TRIS base, TRIS hydrochloride, EDTA, Bromophenol Blue, Xylencyanol FF - GHS07 Warning - - SCH m_en 28/05/15 Review 04 Page 1/12 SCH m_en 28/05/15 Review 04 Page 2/12
MATERIALS REQUIRED BUT NOT PROVIDED IN THE KIT - Laminar airflow hood. - Disposable latex gloves or similar material. - Bench microcentrifuge (12,000-14,000 RPM). - Sterile micropipettes and tips with aerosol filter or positive displacement (2-20 µl, 5-50 µl). - Sterile bidistilled water. - Sterile test tubes for microcentrifuge (1.5 ml). - Electrophoresis tank. - Direct current power supply unit (from 50 V to 150 V). - UV transilluminator (from 320 nm to 380 nm). - Photographic equipment or image acquisition system for recording the results. - Ethidium bromide ( 0,05%). OTHER PRODUCTS REQUIRED The reagents for amplification of the DNA or the cdna obtained from the samples to be analysed, for the Positive Control of amplification and for the internal suitability test are not included in this kit. To perform these analytical steps the following accessory products, manufactured by ELITechGroup S.p.A., are recommended: «OligoMIX Alert kit» series, kit for amplification of the DNA or cdna obtained from samples. «Positive Control» series, positive amplification control of plasmid DNA. «DECK» (code DK100), internal suitability test which uses the human beta globin gene as the target. «RECK» (code RK100), internal suitability test which uses the genome RNA of MS2 phage as the target. This kit is exclusively for in vitro use. WARNINGS AND PRECAUTIONS Warnings and general precautions Handle and dispose of all biological samples as if they were capable of transmitting infective agents. Avoid direct contact with the biological samples. Avoid splashing or spraying. The materials that come into contact with biological samples must be treated with 3% sodium hypochlorite for at least 30 minutes or autoclaved at 121 C for one hour before disposal. Handle and dispose of all reagents and all assay materials as if they were capable of transmitting infective agents. Avoid direct contact with the reagents. Avoid splashing or spraying. Waste must be treated and disposed of in compliance with the appropriate safety standards. Disposable combustible materials must be incinerated. Liquid waste containing acids or bases must be neutralised before disposal. Wear suitable protective clothing and gloves and protect eyes / face. Never pipette solutions by mouth. Do not eat, drink, smoke or apply cosmetic kits in the work areas. Wash hands carefully after handling samples and reagents. Dispose of leftover reagents and waste in compliance with regulations in force. Read all the instructions provided with the kit before running the assay. Follow the instructions provided with the kit while running the assay. Do not use the kit after the expiry date. Only use the reagents provided in the kit and those recommended by the manufacturer. Do not mix reagents from different batches. Do not use reagents from other manufacturers' kits. Warnings and precautions for molecular biology Molecular biology procedures, such as extraction, reverse transcription, amplification and detection of nucleic acids, require qualified staff to prevent the risk of erroneous results, especially due to degradation of the nucleic acids contained in the samples or due to sample contamination by amplification products. It is necessary to have separate areas for the extraction/preparation of amplification reactions and for the amplification/detection of amplification products. Never introduce an amplification product in the area designed for extraction/preparation of amplification reactions. It is necessary to have lab coats, gloves and tools which are exclusively employed in the extraction/preparation of amplification reactions and for the amplification/detection of amplification products. Never transfer lab coats, gloves or tools from the area designed for the amplification/detection of amplification products to the area designed for the extraction/preparation of the amplification reactions. The samples must be exclusively employed for this type of analysis. Samples must be handled under a laminar flow hood. Test tubes containing different samples must never be opened at the same time. Pipettes used to handle samples must be exclusively employed for this specific purpose. The pipettes must be of the positive displacement type or be used with aerosol filter tips. The tips employed must be sterile, free from DNases and RNases, free from DNA and RNA. Reagents must be handled under a laminar flow hood. The reagents required for amplification must be prepared in such a way that they can be used in a single session. The pipettes employed to handle the reagents must be used exclusively for this purpose. The pipettes must be of the positive displacement type or be used with aerosol filter tips. The tips employed must be sterile, free from DNases and RNases, free from DNA and RNA. Amplification products must be handled in such a way as to reduce dispersion into the environment as much as possible, in order to avoid the possibility of contamination. Pipettes used to handle amplification products must be employed exclusively for this specific purpose. Warnings and precautions specific to components The following component of the contains hazardous reagents. European Community Risk and Safety phrases and GHS Hazard and Precautions phrases applied to this component are listed below. Please, note that hazard labeling is not necessary if quantity per bottle is below 125 g or 125 ml. Electrophoresis Buffer 50x Warning GHS07 H315: Causes skin irritation. H319: Causes serious eye irritation. P264: Wash hands thoroughly after handling. P280: Indossare guanti/indumenti protettivi/proteggere gli occhi/proteggere il viso. P302+P352: IF ON SKIN: Wash with plenty of soap and water. P305+P351+P338: IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses if present and easy to do. Continue rinsing. P321: Specific treatment. P332+P313: If skin irritation occurs: Get medical advice/attention. P337+P313: If eye irritation persists get medical advice/attention. P362+P364: Take off contaminated clothing and wash it before reuse. Samples SAMPLES AND CONTROLS The material used with this kit must consist of DNA produced in an amplification reaction. The dimensions of the DNA produced in the amplification reaction must be between 1000 bp and 50 bp to enable identification with this kit. SCH m_en 28/05/15 Review 04 Page 3/12 SCH m_en 28/05/15 Review 04 Page 4/12
Detection controls It is absolutely mandatory to validate each detection session by making the HinfI Marker migrate to the agarose gel, used as a positive detection control. It is absolutely mandatory to validate the specificity of the product of the amplification reaction of a sample by comparing its migration on agarose gel with the migration of the HinfI Marker, used as a molecular weight standard, and with the migration of the amplification reaction product of the positive amplification control. PROCEDURE Preparation of the electrophoresis buffer 1x Before starting the detection phase, prepare the volume of electrophoresis buffer 1x necessary for the electrophoresis detection session, according to the following instructions. Dilute the electrophoresis buffer 50x 50 times in sterile bidistilled water to obtain electrophoresis buffer 1x. For example, transfer 10 ml of electrophoresis buffer 50x to a sterile container measuring 500 ml containing 490 ml sterile bidistilled water. The electrophoresis buffer 1x can be kept at ambient temperature for a maximum of three months. Preparation of the detection session 1 well is required for each sample. It is possible to perform 64 detections, including molecular weight standards and controls. The minimum number of detections per session is 4, including molecular weight standards and controls. Before starting the extraction session it is important to do the following: - referring to the instrument documentation, check the characteristics of the DC power supply unit and the regulations for its safe use by the operator; - referring to the instrument documentation, check the characteristics of the UV transilluminator and the regulations for its safe use by the operator; - define the position of the molecular weight standards, the controls and the samples in the wells of gel and note these down on the Work Sheet. The Work Sheet must be followed carefully during the transfer of the samples into the wells and will be used for correct interpretation of the results. Preparation of the electrophoresis apparatus 1. Make sure that the power supply unit is switched off and that the electrodes are disconnected from the electrophoresis tank. 2. Remove one plastic tray containing a 8-well Ready Gel and remove the protection from the two adhesive strips on the bottom of the tray. 3. Remove the lid which seals the upper part of the tray with the 8-well Ready Gel (Attention: 8-well Ready Gel contains ethidium bromide). 4. Place the tray with the 8-well Ready Gel in the empty electrophoresis tank, attaching it with the adhesive strips and positioning it so that the wells are on the same side as the negative electrode (black). N.B.: For optimal electrophoretic separation, the 8-well Ready Gel tray must be perfectly parallel to the length of the electrophoretic tank. 5. Fill the electrophoretic chamber with the electrophoresis buffer 1x to more than 5 mm above the rim of the tray containing the 8-well Ready Gel so that the electric current can be sent through the gel. Preparation of the sample N.B.: If the «DECK» or «RECK» internal suitability tests have been used in the amplification reaction, the samples must be transferred straight to the wells of gel without adding loading buffer 6x. Follow the procedure described below from point 9. 6. Remove a 1.5 ml microcentrifuge test tube (not supplied with kit) for each sample that is to be analysed, including the amplification controls and one for the molecular weight standards, and mark them clearly using a permanent marker pen 7. Transfer 3 µl of the loading buffer 6x to the 1.5 ml microcentrifuge test tubes for the samples to be analysed including the amplification controls. 8. Remove 15 µl of the amplification product and transfer to the 1.5 ml microcentrifuge test tubes with the loading buffer 6x. Mix well and pipette the volume of 15 µl three times into the loading buffer 6x. Repeat this operation for each sample including the amplification controls. 9. Transfer 10 µl of HinfI Marker to the 1.5 ml microcentrifuge test tubes for molecular weight standards. Execution of electrophoretic separation 10. Make sure that the power supply unit is switched off and that the electrodes are disconnected from the electrophoresis tank. 11. Transfer 18 µl of each sample prepared (or directly of the amplification product when using «DECK» or «RECK») to a well of 8-well Ready Gel, as previously noted on the Work Sheet. N.B.: Carefully transfer the prepared sample to the wells so that it deposits on the bottom of the well and does not overflow into the next wells. 12. Transfer 10 µl of HinfI Marker into the well, as previously noted on the Work Sheet. 13. Close the electrophoresis tank using the safety lid and connect the electrodes: - negative electrode (black) on the same side as the gel with the wells; - positive electrode (red) on the side opposite the gel where DNA is to migrate. N.B.: During these operations, be careful not to jolt the electrophoresis tank, to prevent the samples from spilling out of the wells. 14. Adjust the electric potential difference on the power supply unit so that the electric field is 6-8 V / cm. For example, if the electrodes on the electrophoresis tank are 15 cm apart, it is recommended to apply an electric potential difference of 100-120 V. 15. Switch on the power supply unit and check to see that the loading buffer 6x dyes migrate towards the positive electrode (red). (Attention: an electric current is circulating in the electrophoresis apparatus. 16. Proceed with electrophoresis separation until the migration front of the blue dye of the loading buffer 6x has reached approx. one third of the length of the 8-well Ready Gel. This usually takes 30-40 minutes. (Attention: an electric current is circulating in the electrophoresis apparatus. 17. Switch off the power supply unit, wait 10 seconds, then disconnect the electrodes and remove the lid of the electrophoresis tank. N.B.: The electrophoresis buffer 1x can be used for a maximum of 4 different electrophoresis detections sessions within two days, kept in a sealed electrophoresis tank. N.B.: The amplification reaction product can contaminate subsequent assays. Isolate residual amplification reaction product, used gel, used electrophoresis buffer 1x and other waste produced in the electrophoresis detection. SCH m_en 28/05/15 Review 04 Page 5/12 SCH m_en 28/05/15 Review 04 Page 6/12
DNA detection 18. Within 30 minutes of the end of electrophoretic separation, remove the tray with the 8-well Ready Gel from the electrophoresis tank, then gently extract the gel from the plastic tray and transfer it to the switched-off UV transilluminator (Attention: 8-well Ready Gel contains ethidium bromide). N.B.: The amplification reaction product can contaminate subsequent assays. Avoid contaminating the environment with the electrophoresis buffer 1x. 19. Switch on the UV transilluminator to view the electrophoretic separation of the DNA in the gel and record the results with photographic equipment or an image acquisition system. (Attention: ultraviolet radiation is hazardous for the eyes and skin. Avoid direct or indirect exposure of the eyes and skin to ultraviolet radiation). N.B.: 8-well Ready Gel contains ethidium bromide, hence no dyeing procedure is normally required to highlight the DNA. If the fluorescent signal is not very visible, it is possible to strengthen it as follows: a) in a tank used specially for this purpose, to 100 ml of electrophoresis buffer 1x add 50 µl of ethidium bromide (not provided with the kit) and mix well; b) submerge the gel for 30 minutes without the plastic tray of the electrophoresis buffer 1x and, if possible, keep it moving gently on a tilt table; c) transfer the gel to the UV transilluminator and follow the procedure from para. 19. Interpreting the results Electrophoretic separation of the HinfI Marker is used to validate the detection session as shown in the following table: HinfI Marker Visible bands Detection CORRECT If the bands of the HinfI Marker are not visible, it means that problems have occurred in the detection phase (gel transfer, migration or fluorescence problems) which may cause negative results. The detection session is invalid and must be repeated. Electrophoretic separation of a sample that is under examination is used to evaluate the presence and estimate the dimensions and hence the specificity of the amplification reaction product. This kit is able to detect a minimum of 5 ng DNA produced by the amplification reaction (detection limit of the product, see paragraph on Performance Characteristics on page 10). The results for each sample are used to detect the DNA product of the amplification reaction, as shown in the following table: Sample Band visible DNA produced by the amplification reaction PRESENT It is absolutely mandatory to validate the specificity of the product of the amplification reaction by comparing its migration to gel with the migration of the molecular weight standards of the HinfI Marker, and with the migration of the amplification reaction product of the positive control. This kit is able to resolve molecules of DNA produced by the amplification reaction of between 1000 bp and 50 bp (separation range of the product, see paragraph on Performance Characteristics on page 10). The results for each sample are used to detect the DNA product of the amplification reaction, as shown in the following table: Migration of the DNA produced by the amplification reaction Correct for the marker and the positive control Incorrect for the marker and the positive control DNA produced by the amplification reaction SPECIFIC NON-SPECIFIC The presence of the specific product of the amplification reaction means that target DNA is present in the starting sample as shown in the following table: DNA produced by the amplification reaction Result of electrophoretic detection Assay result Target DNA in the starting sample PRESENT and SPECIFIC positive positive PRESENT PRESENT but NON-SPECIFIC negative negative NOT DETECTED NOT DETECTED negative negative NOT DETECTED The presence of non-specific amplification reaction products has no significance for the detection of target DNA in the starting sample. The significance of the availability of target DNA in the starting sample is described in the instructions manual for the products in the «OligoMIX Alert kit» series. Band not visible NOT DETECTED If the result of the electrophoretic separation of a sample is Not Detected, this does not necessarily mean that the DNA produced by the amplification reaction is not present at levels lower than the detection limit for the product (see paragraph on Performance Characteristics on page 10). In this case the result would be a false negative. SCH m_en 28/05/15 Review 04 Page 7/12 SCH m_en 28/05/15 Review 04 Page 8/12
By way of example, below is a diagram representing the electrophoretic separation of a detection session for the amplification reaction products of 5 samples. In this example the dimensions of the amplification reaction product for the parameter we are concerned with are 110 bp and the dimensions of the amplification reaction product of the internal suitability test «DECK» or «RECK» are 600 bp approx. Wells 1380 bp Molecular weights Positive control Negative control Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 517 bp * * * * 456 bp 600 bp* 600 bp* 600 bp* 600 bp* 396 bp 247 bp 75 bp 110 bp 110 bp 110 bp 110 bp 65 bp 46 bp 250 bp * Using the «DECK» or «RECK» systems, the negative samples of the amplification reaction product for the parameter concerned present, if suitable, the amplification product of the internal suitability test. Furthermore, with the «DECK» or the «RECK» system, the positive samples of the amplification product for the parameters concerned may also present the amplification product of the internal suitability test. Key to the diagram: - Molecular weights, the HinfI Marker has bands of between 1380 bp and 46 bp. - Positive amplification control, the specific amplification product for the parameter concerned is present at 110 bp. - Negative amplification control, no amplification product is present. - Samples 1 and 2, positive samples, the specific amplification product for the parameter concerned and the specific amplification product for the internal suitability test are present. - Sample 3, sample in which the specific amplification product for the parameter concerned is not detected while the specific amplification product for the internal suitability test is present. - Sample 4, positive sample, the specific amplification product for the parameter concerned is present (the specific amplification product for the internal suitability test is absent in this case). - Sample 5, sample in which the specific amplification product for the parameter concerned is not detected (a non-specific amplification product is present) and the specific amplification product for the internal suitability test is present. PROCEDURE LIMITATIONS Only use DNA produced from the amplification reactions of nucleic acids with this product. The results obtained with this product are subject to the correct collection, transport, storage, preparation, extraction and amplification of samples. To avoid result errors it is therefore necessary to take particular care during these phases and to carefully follow the instructions provided. This product must be handled by personnel trained in the processing of chemical preparations to prevent accidents with potentially serious consequences for the user and other persons. This product requires the use of work clothes and premises that are suitable for the processing of chemical preparations to prevent accidents with potentially serious consequences for the user and other persons. This product must be handled by personnel trained in molecular biology techniques, such as extraction, amplification and detection of nucleic acids, to avoid false positives in subsequent stages of the analyses with potentially serious consequences for the patient. This product must be handled in separate areas for extraction/preparation of the amplification reactions and for amplification/detection of amplification products to avoid false positive results in subsequent stages of the analyses with potentially serious consequences for the patient. This product requires the use of special clothing and instruments for extraction/preparation of amplification reactions and for amplification/detection of amplification products to avoid false positive results in subsequent stages of the analyses with potentially serious consequences for the patient. A negative result obtained with this product means that the DNA produced by the amplification reaction has not been detected in the electrophoretic separation, however, this does not necessarily mean that DNA is not present at levels lower than the detection limit for the product (see paragraph on Performance Characteristics on page 10). In this case the result is a false negative. As with any diagnostic device, there is a residual risk of obtaining false negatives with this product. This risk cannot be eliminated or reduced any further. In particular situations, this residual risk can contribute to incorrect decisions with potentially grave consequences for the patient. Analytical sensitivity: detection limit PERFORMANCE CHARACTERISTICS The analytical sensitivity of this detection system, in terms of the detection limit, allows us to verify the presence of approx. 5 ng of DNA in the amplification reaction product. The analytical sensitivity was checked using plasmid DNA as the calibrated reference material, whose initial concentration was measured by spectrophotometer. Plasmid DNA was digested with the HinfI restriction enzyme which causes various fragments among which, one fragment measuring 247 bp, or 7.7% of the initial DNA, and one fragment measuring 75 bp, or 2.3% of the initial DNA. A sample with a concentration of 100 ng of plasmid DNA in 10 µl was used in 2 repeats in different sessions to perform the detection procedure. In all the repeats of the sample, the fragment measuring 247 bp was visible in quantities of 7.7 ng and the fragment measuring 75 bp was visible in quantities of 2.3 ng. SCH m_en 28/05/15 Review 04 Page 9/12 SCH m_en 28/05/15 Review 04 Page 10/12
Analytical sensitivity: separation range The analytical sensitivity of this detection system, in terms of the separation range, allows us to resolve molecules of DNA measuring between 1000 bp and 50 bp in the amplification reaction product. The analytical sensitivity was checked using plasmid DNA as the calibrated reference material, whose initial concentration was measured by spectrophotometer. Plasmid DNA was digested with the HinfI restriction enzyme which causes various fragments whose dimensions are listed below: 1380 bp, 517 bp, 459 bp, 387 bp, 247 bp, 75 bp, 65 bp, 46 bp, 22 bp. A sample with a concentration of 1 µg of plasmid DNA in 10 µl was used in 2 repeats in different sessions to perform the detection procedure. In all the repeats of the sample, the fragments measuring from 1380 bp to 46 bp were resolved and identifiable. N.B.: The complete data and results of the tests carried out to evaluate the performance characteristics of the product are recorded in Section 7 of the Product Technical File "ELECTROPHORESIS", FTP EPH. Catalogue number. Temperature limits. Batch code. Use by (last day of month). SYMBOLS REFERENCES T. Maniatis et al. (1987) Molecular cloning: a laboratory manual Cold Spring Harbor, NY: chapter 6 Signal very faint or absent with the marker Possible causes Error in the layout of gel in the electrophoresis tank. Error in the arrangement of the electrodes in the electrophoresis tank or in the power supply unit. Error dispensing marker in gel. TROUBLESHOOTING Solutions The wells must be on the same side of the electrophoresis tank as the negative electrode (black). Take care when connecting up the electrodes. Carefully transfer the marker to the wells so that it deposits on the bottom. CONT In vitro diagnostic medical device. In keeping with the requirements of European Directive 98\79\EC for in vitro diagnostic medical devices. Contents sufficient for "N" tests. Contents. Please refer to the instructions for use. Keep away from sunlight. Marker degradation. Power unit setting error. Dilution error in the electrophoresis buffer. Electrophoretic separation too long. Tray present on the UV transilluminator. Degradation of the ethidium bromide in the gel. Use a new aliquot of marker. Check the electric potential difference setting on the power supply unit. Check dilution of the electrophoresis buffer. Interrupt migration when the blue dye of the loading buffer has reached one third of the length of the gel. Remove the gel from the plastic tray before transferring it the UV transilluminator. Re-dye the gel as described in the Procedure section. Manufacturer. The purchase of this product allows the purchaser to use it for detection of amplified nucleic acid sequences for a licensed diagnostic parameters providing human in vitro diagnostic services. This right is granted only if this product is used in association with ELITechGroup S.p.A. licensed products for "Positive Control" and for amplification. No general patent or other license of any kind other then this specific right of use from purchase is granted hereby. SCH m_en 28/05/15 Review 04 Page 11/12 SCH m_en 28/05/15 Review 04 Page 12/12