Traditional Thermal Cycler Validation Kit
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1 (en) Order No Traditional Thermal Cycler Validation Kit for conventional PCR block cyclers version 1.0 Instructions for Use Reagents for 2 applications Manufacturer: Minerva Biolabs GmbH, Koepenicker Strasse 325, Berlin, Germany FOR USE IN RESEARCH AND QUALITY CONTROL
2 Symbols Lot No. Order No. Expiry date Store at Contains reagents for 2 applications Manufacturer
3 INDICATION Verification of block heated PCR cyclers by Installation Qualification (IQ), Operational Qualification (OQ) and Performance Qualification (PQ) according to legal requirements, like ISO 17025, EN 45001, ISO 13485, ISO/TS 20836:2007, GLP, GMP and others. EXPLANATION OF THE TEST False negative PCR results or unspecific amplifications might be caused by a defect PCR cycler. Both events are highly critical for Good Laboratory Practise. Verification of the correct temperature control of the used equipment is usually a stressful issue. To comply with quality management systems is not easy to fulfil for PCR cyclers. Electronic temperature sensors offered for purchase or as service usually measure the temperature homogeneity in a cycler block, only, which does not necessarily reflect all parameters which need to be controlled by the cycler and can fail to provide reliable PCR results. Only a reference setup can investigate all relevant parameters of the process reliably. TEST PRINCIPLE The Traditional Thermal Cycler Qualification Kit provides temperature sensitive PCR reactions to monitor an upper and lower temperature range in one run. The primer sequences in combination with a regular PCR protocol were designed to react extremely sensitive to incorrect temperature control, temperature homogeneity, precision and timing. Amplification will be altered and indicated with different band pattern at temperature differences of more than 2 C. The cycler performance is tested at standard PCR settings to reflect most users applications. In addition, the preadjusted target concentrations are only amplified at high PCR efficiencies as an additional indicator for accurate temperature control of the thermal cycler. last update
4 REAGENTS Each kit contains reagents for 2 runs. The expiry date of the unopened package is marked on the package label. The kit components are stored until use at +2 to +8 C. The lot specific Certificate of Analysis can be downloaded from our website ( Kit Component Qualification Tubes Caps Rehydration Buffer Marker Quantity 6 strips, 8 vials each, dried 6 strips, domed 1 x 1.6 ml 1 x 50 μl NEEDED, BUT NOT INCLUDED IN THE KIT The Traditional Thermal Cycler Qualification Kit contains all reagents required to perform two runs on one cycler or one run on two individual block heated cyclers equipped with holes for 0.2 ml PCR tubes. For other formats the reaction mix needs to be transferred into to corresponding PCR tube (e.g. 0.5 ml tubes), which are not supplied with the kit. Also, a 100 μl pipette with corresponding filter tips to dispense the rehydration buffer is needed. PRECAUTIONS For use in research and quality control. This kit should be used only by trained persons. This kit does not contain hazardous substances and may be disposed of according to local regulations. TEST PROCEDURE The validation should be precisely performed as described: 1. Rehydration of the reagents 1. Centrifuge or tip the Qualification Tubes on the table to collect the lyophilized material at 2. Peel off protective film from Qualification Tubes. 3. Aliquot 25 μl of the Rehydration Buffer into each PCR reaction tube. Close the tube tightly. 4. Incubate 5 min at room temperature 5. Vortex briefly and spin for 5 sec After reconstitution, the reagents should be used immediately. 2 last update
5 2. Loading the Qualification Tubes The loading scheme depends on the block format of the PCR cycler. The following schemes are recommended for regular testing. If particular peltier elements, segments of the block or cavities are already subject of investigation, the strips or even individual tubes can be placed flexible within the block. 96 well block: 48 well block: 24 well block: last update
6 3. Starting the reaction Edit and start the following cycler program. The programming process of your cycler is explained in the manual of the instrument: 1 cycle 94 C for 2 min 35 cycles 94 C for 30 sec 67 C for 30 sec 72 C for 30 sec cool down 4 C to 8 C 4. Result reading Prepare a 1.5% standard agarose gel, approx. 5 mm thick, with a 5 mm-comb. Load 5 μl of each PCR reaction and of the marker provided with the kit in each lane. No loading buffer and running dye are required. Stop electrophoresis after 2 cm run distance (depending on the electrophoresis chamber used e.g. run for 20 minutes at 100 V). INTERPRETATION OF RESULTS The cycler is passing the test and the results of the check are valid if one band is visible (Fig. 1). If the results are valid but the cycler does not comply with the expected specifications either no band or two bands are visible: Fragment size Interpretation 144 bp and 210 bp Lower temperature range failure annealing temperature failure denaturation temperature ok 144 bp Setup check ok no bands Lower temperature range failure annealing temperature failure denaturation temperature failure If none of the bands are visible in all reactions the experiment should be repeated to exclude a setup mistake. For the re-run the annealing temperature should be reduced from 67 to 64 C to provoke amplification. If the re-run does not show amplification products and if the cycler is already under suspect for abnormal routine PCR results the device should by send in for service. If two bands are visible either the setup of the test was not ok or the thermal cycler shows a fatal error. Please note, that all PCR reactions should show a uniform result. If not, most likely one or even more of the Peltier elements show a malfunction. In this case the experiment should be repeated with an adopted loading scheme. 4 last update
7 Fig. 1: Gel figure showing results obtained at different annealing temperatures A: Marker B: Temperature too low. C: Temperature correct. D: Temperature too high. A B C D NOTES ON THE TEST PROCEDURE 1. This leaflet must be widely understood for a successful use of the Thermal Cycler Validation Kit. The reagents supplied should not be mixed with reagents from different lots and used as an integral unit. The reagents of the kit should not be used beyond its shelf life. 2. Any deviation from the test method can affect the results. 3. The use of control samples is not required as the PCR set contains a setup check. Appendix Limited Product Warranty This warranty limits our liability for replacement of this product. No warranties of any kind, express or implied, including, without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided. Minerva Biolabs shall have no liability for any direct, indirect, consequential, or incidental damages arising out of the use, the results of use, or the inability to use this product. last update
8 Related Products MB Taq DNA Polymerase /-0100/-0200/-0250 MB Taq DNA Polymerase (5 U/μl) 50/100/200/250 units /-1100/-1200/-1250 MB Taq DNA Polymerase (1 U/μl) 50/100/200/250 units Diagnostic Kits for conventional PCR /-1050/-1100/-1250 Venor GeM Classic Mycoplasma Detection Kit 25/50/100/250 tests /-7048/-7096/-7240 Venor GeM Advance Mycoplasma Detection Kit 24/48/96/240 tests /-8050/-8100/-8250 Venor GeM OneStep Mycoplasma Detection Kit 25/50/100/250 tests /-1050/-1100/-1250 Onar Bacteria Detection Kit 25/50/100/250 tests Diagnostic Kits for qpcr SMB /-1006 Venor GeM Prime Mycoplasma Detection Kit* 25/100 tests SMB /-1004 Intego Mycoplasma Detection Kit* 25/100 tests SMB /-1002 Microsart AMP Mycoplasma* 25/100 tests Mycoplasma Elimination /0500/1000 Mynox Mycoplasma Elimination Reagent 2/5/10 treatments /0501/1001 Mynox Gold Mycoplasma Elimination Reagent 2/5/10 treatments Quantification Standards, 100 μl each, 1x10 6 genomes/μl Acholeplasma laidlawii Mycoplasma arginini Mycoplasma fermentans Mycoplasma hyorhinis Mycoplasma orale Mycoplasma pneumoniae Mycoplasma synoviae Spiroplasma citri 10CFU TM Sensitivity Standards,3 vials with 10 CFU each, 2 vials negative control Mycoplasma arginini Mycoplasma orale Mycoplasma gallisepticum Mycoplasma pneumoniae Mycoplasma synoviae Mycoplasma fermentans Mycoplasma hyorhinis Acholeplasma laidlawii Spiroplasma citri Mycoplasma Set, all EP listed species 2 vials per species, 10 CFU each DNA Remover TM DNA Decontamination Reagent, spray bottle 250 ml DNA Decontamination Reagent, refill bottles 4 x 500 ml Mycoplasma Off Surface Disinfectant Spray, spray bottle 1000 ml Surface Disinfectant Spray, refill bottles 5 x 1000 ml Mycoplasma Off-Wipes Surface disinfectant Wipes in dispenser box 120 wipes Surface Disinfectant Wipes, refill pack 5 x 120 wipes ZellShield T /-0150 Contamination Prevention Reagent 100x concentrate 1000 ml/ 5 x 1000 ml * Products distributed by Sartorius Lab Products and Services.
9 Manufacturer Minerva Biolabs GmbH Koepenicker Str. 325 D Berlin Germany Ordering Tel. +49 (0) Fax +49 (0) Product Information Technical Service Tel. +49 (0) Made in Germany Minerva Biolabs GmbH develops and manufactures products in accordance with EN ISO 9001:2008 and EN ISO 13485:2003 quality system requirement. Reg.No. SY & SX as precise as diagnostics should be TM
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