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1. inhibitors present Test No amplification Potential inhibitors present EEE area with sterile water repeat 187bp ve Contamination is present Isolate and clean pipette used Clean contaminated area with solution Same ipe Test designed to remove DNA Repeat test No amplification Wipe test negative No contamination 10 Quality Assurance and Control Assay testing PCR amplicon from a Biofortuna Kit was allowed to dry on a solid surface The wipe 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 Genomic DNA was allowed to dry on a solid surface The wipe test was performed on gDNA at 100ng wul and then in dilutions from 1x10 to 1x10 The DNA was detected using gDNA with concentrations ranging from 0 1ng ul to 100ng ul 11 References 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 Page 7 of 9 Dot123v1 USA English Instructions for Use for Biofortuna SSPGo HLA Wipe Test BF 40 01 12 HLA Wipe Test Sample record sheet Sample Record Sheet It is recommended that this sample record sheet is photocopied prior to use as the Wipe Test Kit has sufficient tests for 36 zones 3 wipe zones per 8 well strip 12 strips per kit Test Date Test performed by Approved By Other information Kit Lot Number Use see Instructions Tes2. BE buffer Transfer a minimum of 5ul 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 Page 6 of 9 Dot123v1 USA English Instructions for Use for Biofortuna SSPGo HLA Wipe Test BF 40 01 9 Interpretation Test results for swabbed zones are only valid if the positive control is positive and the negative control is negative Tests for swabbed zones are only valid if the corresponding inhibition control test is positive A 187bp amplicon should be observed if there is 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 Rx Dye Use Result Conclusion Acti SSS O 187bp ve Test valid DNA suitable PCR Positive effective control No amplification Test invalid Repeat entire assay with different DNA control 187bp ve Test invalid Water and or pipette Test with different water and pipette Negative 2 Purple contaminated FA Control No amplification Test valid Water not contaminated Inhibition 187bp ve No
3. Dot123v1 USA English Instructions for Use for Biofortuna SSPGo HLA Wipe Test BF 40 01 teal BIOFORTUNA SSPGo Instructions for Use Biofortuna SSPGo HLA Wipe Test BF 40 01 USA version 1 May 2014 Page 1 of 9 Dot123v1 USA English Instructions for Use for Biofortuna SSPGo HLA Wipe Test BF 40 01 iia i 1 Intended Use The Biofortuna SSPGo HLA Wipe Test is intended to be used as a Quality Control test to monitor laboratory areas and equipment for PCR amplicon contamination that may be generated by the use of Biofortuna SSPGo products 2 Introduction 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 3 Test Description Each SSPGo HLA Wipe Test consists of a strip of eight PCR wells containing freeze dried PCR buffer polymerase and primers specific for the HLA DRA gene and will produce an 187bp amplicon from human genomic DNA Since all Biofortuna kits utilise a DRA amplicon as an internal control any contamination from use of Biofortuna SSPGo kits will have amplification of the DRA gene and will be detected by the wipe test primers Each strip of eight wells tests up to thr
4. See Note 1 and Note 2 for additional information Use only the sealing sheets or caps provided Ensure PCR wells are sealed tightly after adding DNA as omitting this may lead to evaporation during PCR amplification Pay particular attention to edges and corners Once you have removed the original caps from the 8 well strips DO NOT re use Only use the additional spare caps provided to carry out the PCR reaction 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 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 sample at a temperature of up to 20 C and humidity of no more than 60 Note 2 Once hydrated with sample 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 Page 4 of 9 Dot123v1 USA English Instructions for Use for Biofortuna SSPGo HLA Wipe Test BF 40 01 i Directions for Use Note A frequent source of contamination is PCR pipettes and it is advisable to swab the tip of the pipette and if possible inside the barrel It is also recommended that all contamination tests are performed with a pipette that has been tested and shown to be negative If contamination is discovered follow your laboratory guidelines to eradicate contamin
5. _ 4 Purple Zone1WipeTest S O 6 Ls e 36 sterile swabs e 12x8 PCR caps e 1x instructions for use and one 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 Page 2 of 9 Dot123v1 USA English Instructions for Use for Biofortuna SSPGo HLA Wipe Test BF 40 01 5 Reagents and Equipment Not Supplied e Appropriate calibrated pipettors and sterile tips e g P10 pipettor with 10ul filter tips e DNA isolation kit equipment e UV spectrophotometer suitable for the measurement of DNA concentration and purity 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 using 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 Immunog
6. ation and retest zones 1 In a DNA free location label a sterile DNase free polypropylene 2ml tube for each of the wipe test zones to be tested Label an extra tube Negative Control 2 Using a known contamination free pipette add 500ul sterile molecular grade distilled water to each of the 2ml tubes Wet a provided sterile plastic applicator swab in each wipe test tube Wipe the zone to be tested with the moistened applicator Snap or cut off the plastic stem of the applicator and place in the original 2ml tube of water Vortex briefly Remove and discard the swab with sterile forceps Centrifuge 1 minute 10 000 to 13 000 rpm in a micro centrifuge to remove particulate matter Open the test foil pouch All tests are to be rehydrated with a total of 10ul of liquid as shown in diagram 1 10 Negative control Add 10ul sterile molecular grade water to the negative control reaction 2 purple 11 Add 5ul of the sterile water to reactions 1 4 6 8 12 Positive control Add 5yl of 10ng ul human genomic DNA to reaction 1 red reaction and reactions 3 5 amp 7 blue 13 Wipe test reactions Add 5ul of centrifuged liquid wipe water from wipe zone 1 to reactions 3 amp 4 from wipe zone 2 to reactions 5 amp 6 and from wipe zone 3 to reactions 7 amp 8 14 All reactions now have 10ul rehydrated volume as shown in diagram 1 Cap the reactions with the provided caps and proceed with the usual SSPGo PCR parameters as s
7. ee wipe test zones for contamination Each strip has integral positive and negative control reactions as well as inhibition control reactions for each wipe test zones To test a zone for contamination it is first wiped with a swab which is then soaked in water This water is used as a template in the wipe test and as a 50 50 mixture with genomic DNA as a PCR inhibition test It is recommended to test for contamination on a regular basis Typical zones to be tested include DNA preparation area PCR setup area and post amplification area Typical items to be tested include work benches pipettes centrifuges refrigerator and freezer handles door knobs and racks Typical solutions to be tested include DNA preparation buffers and DNA diluents Multi use shared reagents such as PCR buffers and Taq polymerase are particularly susceptible to contamination but these do not affect Biofortuna kits since Biofortuna products are complete and only require the addition of DNA 4 Kit Contents e 12 strips of 8 PCR wells each containing pre dispensed freeze dried primers polymerase dNTPs and buffer Each foil packed strip is intended for testing three zones for contamination The eight reaction strip format is shown below Positive Control DNA Negative Control water used to wet swab Blue Zone 1 Inhibition Test 50 DNA 50 wipe water Blue _ Zone 2 Inhibition Test 50 DNA 50 wipe water Blue Zone 3 Inhibition Test 50 DNA 50 wipe water _
8. enetics 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 the test results ap f 6 Safety and Warnings 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 Page 3 of 9 Dot123v1 USA English Instructions for Use for Biofortuna SSPGo HLA Wipe Test BF 40 01 A 7 Storage and Stability Biofortuna SSPGo HLA Typing kits should be stored at 2 28 C 36 82 F Once PCR strips or plates are removed from the foil pouches the reagents should be re hydrated with test sample promptly
9. hown below OON AMAR WwW Diagram 1 a Reaction 3 Reaction 4 Reaction 5 Reaction 6 Reaction 7 Reaction 8 Reaction 1 Reaction 2 RED PURPLE BLUE PURPLE BLUE PURPLE BLUE PURPLE 5ul Sterile 10ul Sterile Zone 1 Zone 1 Zone 2 Zone 2 Zone 3 Zone 3 Water Water 5ul Wipe 5ul Wipe 5ul Wipe 5ul Wipe 5ul Wipe 5ul Wipe 5ul DNA Water Water Water Water Water Water 5ul DNA 5ul Sterile 5ul DNA 5ul Sterile 5ul DNA 5ul Sterile Water Water Water s gt Control Tests Wipe Tests 3 Locations RE HYDRATION NOTE Once PCR wells are removed from the foil pouches the reagents should be re hydrated with DNA promptly See Note 1 and Note 2 for additional information 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 20 C and humidity of no more than 60 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 Ensure the final DNA sample does not contain more than 2 5mM Tris 0 25mM EDTA Only use DNA extracted from citrate and EDTA collected samples As heparin may inhibit PCR it is recommended that DNA should not be extracted from heparinised blood samples Hemoglobin has been shown to interfere with SSPGo HLA kits when present in DNA samples at greater than 1 mg dL Page 5o0f 9 Dot123v1 USA English Instructions for Use for Biofort
10. t Result kua Sample Information E a a ff Zone 1 Inhibition Test Zone 1 Wipe Test Zone 2 Inhibition Test Zone 2 Wipe Test Zone 3 Inhibition Test Zone 3 Wipe Test Page 8 of 9 Dot123v1 USA English Instructions for Use for Biofortuna SSPGo HLA Wipe Test BF 40 01 13 Guide to Symbols Used Contains sufficient for N y Lot Number o eidem Consult Instructions for Global Trade Item Number 14 Manufacturer Contact Details Biofortuna Ltd a 1 Hawkshead Road Croft Business Park ep Bromborough CH62 3RJ UK T 44 0 151 334 0182 E info biofortuna com BIOFORTUNA W www biofortuna com IMPI Page 9 of 9
11. una SSPGo HLA Wipe Test BF 40 01 DNA can be extracted using traditional extraction methods Ensure that the OD2 0 289 of the DNA sample falls between 1 66 and 1 94 as measured by UV spectrophotometry 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 1 C per second and enable the heated lid Please refer to the thermal cycler manufacturer s user manual for full instructions for use Thermal cyclers 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 30seconds Denature 96 C 15 seconds Anneal 64 C 50seconds 20 cycles Extend 72 C 30seconds 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 concentration 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 T
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