e-Myco VALiD-Q User Manual
Contents
1. hyorhinis v U Instruction Manual STORAGE AND STABILTY e Storage condition Store the product at 25 15 C after receiving e Expiration e Myco VALID Q Mycoplasma PCR Detection Kit can be stored for up to 12 months without showing any reduction in performance and quality under appropriates or age T condition The expiration date is labeled on the product box J Crude lysate Purified DNA Supernatant urified 2X qPCR Detection Template PCR tube Master Mix Solution optic grade APPLICATION The kit is used for the detection of mycoplasma species that are most commonly encountered in cell culture including M peumoniae M arginini M fermentans M hyorhinis M orale and A laidlawii Perform Ei Siep Temp Channel seking Furthermore this kit can detect other various species of mycoplasma Real time PCR Seed Mycoplasma FAM E n PCR and Signal Detection MATERIALS REQUIRED BUT NOT PROVIDED e Realtime PCR Instrument e G spin total Extraction Kit Cat 17045 Analyze the results e Pipettes e Sterile pipette tip with filter e Centrifuge for micro centrifuge tubes e Vortex mixer e Disposable gloves gt SA N t RO N Wal f e SSZ Biotechnology PROTOCOL DATA VALIDATION You can use this protocol just for detecting the contamination of mycoplasma However if you want to perform genotyping for the detailed determination of species please purify the genomic DNA of suspected Mycoplasma infected cells2. 5yl of purified genomic DNA as a template using the G spin Total DNA Extraction Kit Cat No 17046 and then resuspend after adding Master Solution 15yl in the PCR tube Note Appropriate amounts of DNA template sample genomic DNA 50 ng 100 ng 2 Follow protocol A from step 10 Note Recommend to perform one negative control reaction by adding Sul of sterile water We recommend to add Sul of control DNA for positive control reaction 1 When the reaction is finished put a cut off value according to the below table Set Manual baseline Threshold Ct Cutoff Value 3 15 Auto Drop after 36 cycle 2 Valid Results Ct value of control should be as below table Items FAM HEX Items FAM HEX Positive Control 18 22 22 25 Negative Control lt 36 lt 36 DATA INTERPRETATION 1 Expected Real time RT PCR Data Mycoplasma IPC No Samples FAM HEX Interpretation 1 Positive control Valid 2 Negative control Valid 3 Test 1 Positive A Test 2 7 Positive High concentration of Mycoplasma DNA Test 3 Negative Mycoplasma Free 6 Positive Control Retest 7 Negative Control Contamination EXPLANATION OF SYMBOLS LOT Batch number EA Sufficient for 50 tests m Manufacturing date TROUBLESHOOTING GUIDE Product number for research use only Storage temperature limitation bs Expire date Observation Possible Cause Recommendation Incorrect
3. cells in 1 ml of sterile PBS or DPBS solution for washing 4 Spin the tube in a microcentrifuge for 10 15 seconds Carefully decant the supernatant Note Option Repeat this wash step once more 5 Resuspend the cell pellets in 100 pl of sterile PBS or DPBS solution Note If you want the best result use of PBS solution is better than Tris 10 mM pH 8 5 TE 10 mM Tris 0 1 mM EDTA or autoclaved DW 6 Heat the samples at 95 C for 10 min and vortex for 5 10 sec Then centrifuge for 2 min at 13 000 rpm with a tabletop centrifuge at RT 7 Transfer an aliquot of the heated supernatant to a fresh tube This supernatant will be used as the template in the PCR 8 Prepare Detection Mix by dispensing components to each real time PCR tube in the following manner Components Master Solution per test 2X qPCR Master Mix Solution 10 ul Mycoplasma Detection Solution 5 ul a e 9 Fill up with the supernatant 5ul and Master Solution 15yl in the PCR tube Note 1 For Negative Control 5ul DNase RNase Free Water Note 2 For Positive Control Sul Positive Control 10 After centrifugation put them into a real time PCR system and process reaction Step Cycle Temp Time Channel setting Pre denaturation 1 95 C 5 30 Mycoplasma FAM PCR and 95 C 15 IPC HEX f f 40 VEEE ARIANA AEA NAELA Signal Detection 58 C 1 Under line means signal detection step PROTOCOL B Using genomic DNA as a template 1 Add
4. dye components chosen Hen Oye componen prior t data a a analysis ach tee es Check that all the correct reagents were ARn lt No Template Reaction component omitted added control ARn and no amplification plot Reaction inhibitor present Repeat with purified template Check technique and equipment to confine contamination Use fresh reagents Repeat with aerosol barrier pipette tip after space cleaning ARn lt No Template control ARn and both reaction show an amplification plot Template contamination of reagents Reset the upper lower value of baseline two cycles lower than Ct Value or repeat with diluted sample Amplification plot dips downwards Ct Value less than 15 amplification signal detected too early Amplification plots is not within the log phase PCR efficiency is poor Re optimization the reaction conditions Less template added than expected Increase sample amount Sample is degraded Evaluate sample integrity Ct value is higher than expected More template added than expected Reduce sample amount Check technique and equipment to confine contamination Use fresh reagents Repeat with aerosol barrier pipette tip after space cleaning Ct value is lower than expected Template contamination of reagents Technical advice 82 505 550 5600 Review date 2015 5 6 ea S T aan Cy NA iaa A Wal l FA kag D ERY Biotechnology
5. Test for the detection of Mycoplasma by qPCR analysis e Myco VALID 0 x Mycoplasma qPCR Detection Kit ooye SZ ave A BACKGROUND INFORMATION PACKAGING INFORMATION AND STORAGE Mycoplasmas are small round or filamentous prokaryotic organisms which are a frequent contaminant of cell cultures Mycoplasma depend on their hosts for many nutrients due to their Contents Storage Amount limited biosynthetic capabilities Up to 30 85 of cell cultures may be contaminated with SAR 500 ul x 1 vial mycoplasmas the main contaminants being the species M orale A laidlawii M arginini and AA OPOR MASIE IMPE SONON mA 10 ul test x 50 tests M hyorhinis Although these mycoplasmas do not usually kill contaminated cells they are difficult to Mycoplasma Detection Solution 25 15 C 250 pl x 1 vial detect and can cause a variety of effects on cultured cells including changes in metabolism growth aa viability and morphology there by altering the phenotypic properties of the host cells Many Positive Control External PC 25 15 C 25 pl x 1 vial methods are available for detection of mycoplasma including isolation in broth agar culture direct DNase RNase Free Water 250 ul x 1 vial or indirect fluorescence staining ELISA immunostaining direct or indirect PCR Among those methods direct PCR is the highly sensitive specific and convenient method when the primer design is optimized The e Myco VALID Q Mycoplasma qPCR Detection Kit is compo
6. f the kit optimization of reaction condition and maximizes PCR specificity and sensitivity through the control of non specific priming e The kit contains all reagents required for real time polymerase chain reaction nucleotides KIT WORKFLOW primer and probes reaction buffer polymerase and controls After rehydration of the components the PCR Master mix is easily prepared by just adding the Inhibition Control DNA and Taq DNA polymerase to the Primer amp Probes e The PCR is initiated with a 5 minute high temperature step to melt all nucleic acids and to activate the polymerase In a successful PCR the presence of mycoplasmas will be indicated by 4 Cultivated Cell a signal in the FAM channel PROTOCOL A PROTOCOL B KIT CONTENTS Cell boiling method Genomic DNA method Contents Composition Ka e Real time PCR Reaction solution pel narve si 2X qPCR Master Mix lt 0 01 dATP dTTP dGTP dCTP amp washing Solution lt 0 01 Hot start PCR enzyme lt 0 01 PCR additive materials e Mycoplasma Detection solution lt 0 001 Primer probe for Mycoplasma lt 0 001 Internal control primer pair lt 0 001 Internal control DNA Detection Solution Boiling 95 C 10min DNA purification using G spin Total DNA Extraction Kit Cat No 17046 DNase RNase Free Water Ultrapure sterilized distilled water JP e Mycoplasma positive control Positive Control External PC lt 0 001 Recombinant DNA contained 16S sequence of M
7. sed of a set of primers and NOTICE probe that are specific for the highly conserved mycoplasma16S rRNA coding region including Manual 1 ea M pneumoniae M argnini M hyorhinis M fermentans M orale and A aidlawii The kit is designed e To prevent contamination of mycoplasma DNA during experimental procedure always wear to detect the presence of mycoplasma that might contaminate biological materials such as cultured gloves during sample preparation and PCR reaction setup cells Also the kit can detect mycoplasma within 90minutes sensitively up to 10CFU ml and e To avoid false positives water used in PCR reactions can be UV irradiated includes internal control for verifying a qPCR run as well as positive control DNA e If no internal positive control signal it shows the problem during PCR process Please re test If there is non specific signal in negative control it could be due to the contamination or over PRINCIPLES used template Please re test with proper amount of template e The real time qPCR quantitative polymerase chain reaction DNA amplification technology SHELF LIFE shows high sensitivity and specificity for direct detection of pathogen antigen iNtRON developed a novel platform technique about primer design called CLP complementary locking e 12 months from manufacturing date primer technology which provides flexibility in Tm melting temperature of primer design for e Within 3 months after opening within expiry date o
8. using our G spin Total DNA Extraction Kit Cat No 17046 You may use simply this protocol or your other general boiling methods TECHNICAL TIP 1 Use clean disposable gloves when performing the assay and make sure that the work area is clean prior to starting the assay setup 2 Keep your reagents and PCR mixture tubes on a cold block during reaction setup 3 Use positive displacement pipettes 4 The amplification and detection areas should be physically separated i e do not use the same bench area to set up the PCR reactions and run your gels PROTOCOL A Using the Boiling Extract Method 1 Prepare cell suspensions from the test cell culture in a 1 5 ml tube Then count cell numbers by general counting methods You need at least 5x104 cells per test Note 1 Harvest adherent cells with trypsin EDTA solution using standard techniques Pipette 1 ml of TE treated adherent cells Generally with suspension cells such as K562 you need not treat with TE solution We recommend that you count the cells You should prepare at least 5x10 cells per test Note 2 Strong mycoplasma infections are detected in as little as 10 100 cells while weak infections require cells over 5 000 50 000 cells You can dilute the template according to the infection rates you suspect 2 Transfer the counted cells over 5x10 cells to a 1 5 ml tube Spin the tube in a microcentrifuge for 10 15 seconds Carefully decant the supernatant 3 Resuspend the
Download Pdf Manuals
Related Search