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1. gene and will be detected by the NTC test primers e 12 strips of 8 PCR reactions each containing pre dispensed freeze dried primers polymerase dNTPs and buffer The eight reaction strip format is shown below eaor bye we ple No Template Control Sample 5 No Template Control Sample 6 No Template Control Sample 7 2 No Template Control Sample 4 No Template Control Sample 8 e 12x8 PCR caps e 1x instructions for use e 1x Certificate of Analysis e The MSDS can be downloaded from the Biofortuna website www biofortuna com If you are unable to download from the website please contact your local distributor CleanAmp dNTPs are licensed from Trilink Biotechnologies Inc for use in Biofortuna SSPGo products C C DOT129v1 Instructions for Use Biofortuna SSPGo HLA No Template Control Kit BF 40 02 Page 3 of 7 e Appropriate calibrated pipettors and sterile tips e g P10 pipettor with 10 l filter tips e DNA isolation kit equipment e UV spectrophotometer e Polypropylene tubes e Sterile molecular grade water e Athermal cycler with the following specifications should be used e 96 well thermal cycler with heated lid with a temperature of 104 C for oil free operation e Ramp rate of 1 0 C sec e Temperature range of 4 0 C to 99 9 C e Temperature accuracy of 0 25 C for the range of 35 C to 99 9 C e Temperature calibration traceable to a reference standard e Program the thermal cycler u2. DOT129v1 Instructions for Use Biofortuna SSPGo HLA No Template Control Kit BF 40 02 Page 1 of 7 hal BIOFORTUNA SSPGo Instructions for Use Biofortuna SSPGo HLA No Template Control Kit BF 40 02 CE revision 2 July 2014 C C DOT129V1 Instructions for Use Biofortuna SSPGo HLA No Template Control Kit BF 40 02 Page 2 of 7 The Biofortuna SSPGo No Template Control NTC Kit contains supplementary tests for PCR amplicon contamination to be used in conjunction with Biofortuna SSPGo kits that do not have an integral NTC PCR is a sensitive technique that is susceptible to contamination with DNA amplicon from a previous PCR Contamination can lead to false positive amplification in subsequent PCRs which can lead to incorrect genotyping PCR amplicon can contaminate reagents and samples as well as laboratory equipment such as pipettes Reagents and equipment should be monitored regularly for signs of contamination The NTC test is used to detect potential PCR or DNA contamination present in the DNA diluent used for a test sample Each SSPGo No Template Control kit consists of 12 strips of 8 PCR reactions containing freeze dried PCR buffer polymerase and primers specific for the HLA DRA gene and will produce an 187bp amplicon from human genomic DNA or amplified DNA All Biofortuna kits utilise a DRA amplicon as an internal control therefore any contamination from the use of Biofortuna SSPGo kits will have co amplification of the DRA
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4. d on gDNA at 100ng ul and then in dilutions from 1x10 to 1x10 The DNA was detected in dilutions up to 1x10 1 Bunce M et al Tissue Antigens 1995 Nov 46 5 355 67 2 Saiki RK et al Nature 1986 Nov 13 19 324 6093 163 6 C C DOT129v1 Instructions for Use Biofortuna SSPGo HLA No Template Control Kit BF 40 02 Page 6 of 7 It is recommended that this sample record sheet is photocopied prior to use as the NTC Kit has sufficient tests for 96 samples twelve strips of eight tests Sample 1 Description Sample 2 Description Sample 3 Description Sample 4 Description Sample 5 Description Sample 6 Description Sample 7 Description Sample 8 Description NTC Sample record DOT129V1 Instructions for Use Biofortuna SSPGo HLA No Template Control Kit BF 40 02 Page 7 of 7 VY Number of Tests jcc re EC Representative Uli Consult Instructions for Use al Site of Manufacture vo z In Vitro Diagnostic Expiry Date YYYY MM DD 2 pe Storage Temperature LOT Lot Number _ Distributed by GTIN Global Trade Item Number Biofortuna Ltd aw 1 Hawkshead Road Croft Business Park Bromborough CH62 3RJ UK Et T 44 0 151 334 0182 E info biofortuna com BIOFORTUNA W www biofortuna com MPLY DIAGNOS FranCaise Traductions disponibles Deutsch bersetzungen verf gbar Espa ol Traducciones disponibles Italiano Traduzioni disponibili esk P eklady k dispozici Danske
5. e reagents should be re hydrated with DNA within 3 hours Refer to packaging for expiration date Do not use products after the printed date Do not use kits if the foil pouch is ripped or perforated or if there is no desiccant bag present Using the sealing sheets or caps provided only ensure PCR vessels are sealed tightly after adding DNA as omitting this may lead to evaporation during PCR amplification Pay particular attention to edges and corners Note 1 If necessary the out of pouch non hydrated PCR plates and strips may be held for up to 3 hours prior to addition of DNA at a temperature of up to 21 C and humidity of no more than 60 C C DOT129v1 Instructions for Use Biofortuna SSPGo HLA No Template Control Kit BF 40 02 Page 4 of 7 Note 2 Once hydrated with DNA PCR strips and plates from freshly opened pouches can be stored for up to 24 hours at 2 8 C before the PCR step provided that the wells are well sealed to avoid evaporation Note A frequent source of contamination is PCR pipettes and it is advisable to use a known contamination free pipette to load sample into PCR reactions including this NTC test 1 Open the foil pouch and label appropriately one strip can be used to test between one and eight samples Once opened the PCR reactions should be used within 3 hours 2 For each DNA sample genotyped add 10ul of the water or diluent used to one of the reactions of the NTC strip 3 If a positive control
6. is required use 10ul of 10ng ul human genomic DNA This will result in a positive test amplicon of 187bp 4 Cap the reactions with the provided caps and proceed with the standard SSPGo PCR parameters as shown below RE SUSPENSION NOTE Ensure the PCR mixes are re suspended with the samples within 3 hours of removing the tray from the foil pouch PCR PLATE STRIP HEIGHT PROFILE NOTE It is recommended that the height profile of plates and strips are equivalent when placed in the same PCR machine Different height profiles can cause poor contact with the PCR machines heated lid This may result in poor or failed PCR amplification PCR Parameters The following PCR parameters should be used Ensure ramp speeds of at least 1 C per second and enable the heated lid Please refer to the thermacycler manufacturer s user manual for full instructions for use Thermalcyclers should be calibrated according to the American Society of Histocompatibility and Immunogenetic ASHI or European Federation of Immunogenetics EFI accreditation rules Denature 94 C 5 minutes Denature 96 C 15 seconds Anneal 66 C 50seconds 10 cycles Extend 72 C 30 seconds Denature 96 C 15 seconds Anneal 64 C 50 seconds 20 cycles Extend 72 C 30 seconds HOLD 15 C Gel Electrophoresis These instructions apply to horizontal agarose gel electrophoresis Prepare a 2 agarose gel in 0 5x TBE buffer When the gel is cooled to about 60 C add ethidium bromide to a final co
7. ncentration of 0 5ug ml Cast gel and insert microtitre format combs e g 12x8 wells with 9mm spacing Once set remove the combs and cover gel in 0 5x TBE buffer Transfer a minimum of 5 ul and a maximum of 10ul from each tray or strip reaction to the corresponding well on the gel noting the position of each reaction A 100bp ladder can be useful to aid size determination Run gel for 20 minutes at 10V cm Refer to your electrophoresis system manufacturer s instructions for use for specific equipment details Gels should be imaged using a UV gel documentation system with UV transilluminator C C DOT129V1 Instructions for Use Biofortuna SSPGo HLA No Template Control Kit BF 40 02 Page 5 of 7 Record the results using the sample record table on page 6 of this IFU A 187bp amplicon will be observed if there is SSPGo PCR contamination or DNA contamination present Any smears or bands of different sizes may also indicate PCR contamination but primer dimer and other primer extension artefacts of less than 100bp should be ignored A positive result for any diluent sample indicates that the genotyping of that sample is invalid and should be repeated with another DNA sample using different reagents Assay testing Using PCR amplicon from a Biofortuna Kit the NTC Test was performed on the amplicon undiluted and then in dilutions from 1x10 to 1x10 The amplicon was detected in dilutions up to and including 1x10 The NTC Test was performe
8. sing the PCR Cycling Parameters in Section 8 below Note For specific thermal cycler information refer to the manufacturer s user manual Thermal cycler should be calibrated according to ASHI American Society of Histocompatibility and Immunogenetics or EFI European Federation of Immunogenetics accreditation rules e Gel electrophoresis reagents agarose 0 5x TBE 1000bp DNA molecular weight marker 10mg ml Ethidium Bromide e Gel electrophoresis equipment gel tanks power supply gel documentation system with UV transilluminator Note any change in the specified conditions such as thermal cycler ramp rates may affect interpretation of the test results e Tests should only be carried out by appropriately trained personnel e Handle all reagents in accordance with Good Laboratory Practice e Keep pre and post PCR areas separate Do not bring any post PCR materials back to the pre PCR area e Biohazard Warning Treat all blood products as potentially infectious e Biohazard Warning Ethidium Bromide is a potential carcinogen If used always wear gloves a laboratory coat and protective eye glasses e Biohazard Warning Take care when using UV sources always wear gloves a laboratory coat and protective eye glasses Never view the UV light source directly e Material Safety Data Sheets are available from www biofortuna com Biofortuna SSPGo kits should be stored at 2 28 C Once PCR vessels are removed from the foil pouches th
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