Mycoplasma PCR Detection Kit - Protocol
Contents
1. Precautions and Disclaimers This kit is designed for research purposes only It is not intended for human or diagnostic use Safety Information Ensure that a suitable lab coat disposable gloves and protective goggles are worn when working with chemicals For more information please consult the appropriate Material Safety Data Sheets MSDSs These are available as convenient PDF files online at www norgenbiotek com Important Notes Prior to Beginning Protocol A variable speed centrifuge should be used for maximum kit performance If a variable speed centrifuge is not available a fixed speed centrifuge can be used however reduced yields may be observed Ensure that all solutions are at room temperature prior to use and that no precipitates have formed If necessary warm the solutions and mix well until the solutions become clear again Prepare a working concentration of the Wash Solution by adding 21 mL of 96 100 ethanol provided by the user to the supplied bottle containing the concentrated Wash Solution This will give a final volume of 30 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added Lysate can be prepared from either cell suspension or monolayer Please ensure that you follow the proper procedure for lysate preparation in Step 1a For the isolation of genomic DNA from Preheat a water bath or heating block to 65 C Isolation Control soC An Isolation Control soC2. Master Mix The Mycoplasma Mastermix contains reagents and enzymes for the specific amplification of a 327 region of the genome In addition Norgen s Mycoplasma PCR Detection Kit contains a second Mastermix the PCR Control Master Mix which can be used to identify possible PCR inhibition and or inadequate isolation via a separate PCR reaction with the use of the provided PCR control PCRC or Isolation Control lsoC respectively This kit is designed to allow for the testing of 24 samples Kit Components Lysis Solution 15 mL Wash Solution 9 mL Elution Buffer 3 mL Bead Tubes Mini Spin Columns Coa Collection Tubes Ooo o a Elution tubes 1 7 mL a Nuclease Free Water 1 25 mL Norgen s DNA Marker 0 1 mL Product Insert 1 IsoC Isolation Control PosC Positive Control The isolation control is a cloned PCR product The positive control is a fragment of Mycoplasma cloned in a plasmid Customer Supplied Reagents and Equipment e Disposable powder free gloves Benchtop microcentrifuge 1 5 mL microcentrifuge tubes 65 C water bath or heating block 96 100 ethanol 70 ethanol Storage Conditions and Product Stability All solutions should be kept tightly sealed and stored at room temperature These reagents should remain stable for at least 1 year in their unopened containers The Mycoplasma 2x PCR Master Mix Control 2x PCR Master Mix Mycoplasma Positive Control PosC and the Isolation Control soC should be
3. is supplied This allows the user to control the DNA isolation procedure For this assay add the Isolation Control IsoC to the lysate during the isolation procedure The Isolation Control IsoC must not be added to the sample material directly Do not freeze and thaw the Isolation Control IsoC more than 2 times The Isolation Control IsoC must be kept on ice at all times during the isolation procedure The PCR components of the Mycoplasma PCR Detection Kit should remain at 20 C until DNA is extracted and ready for PCR amplification It is important to work quickly during this procedure 1 Lysate Preparation a k From suspension Centrifuge 15 50 mL of cells growing in suspension for 5 minutes at 3000 x g in order to collect the cell pellet Decant the supernatant and resuspend the cell pellet with PBS or fresh media to obtain the appropriate number of cells 1 x 10 cells From monolayer After trypsinization collect and wash the cells with PBS or fresh media to obtain the appropriate number of cells 1 x 10 cells Centrifuge at 14 000 x g 14 000 RPM for 1 minute to pellet the cells Pour off the supernatant carefully so as not to disturb or dislodge the cell pellet Add 500 uL of Lysis Solution to the cell pellet Resuspend the cells by gentle vortexing Transfer the mixture to a provided Bead Tube and secure the tube horizontally on a flat bed vortex pad with tape or in any commercially available bead beat
4. kept tightly sealed and stored at 20 C for up to 1 year without showing any reduction in performance Repeated thawing and freezing gt 2 x should be avoided as this may reduce the sensitivity If the reagents are to be used only intermittently they should be frozen in aliquots General Precautions The user should exercise the following precautions when using the kit e Use sterile pipette tips with filters e Store and extract positive material specimens controls and amplicons separately from all other reagents and add it to the reaction mix in a spatially separated facility e Thaw all components thoroughly at room temperature before starting an assay e When thawed mix the components and centrifuge briefly e Work quickly on ice Quality Control In accordance with Norgen s ISO 9001 and ISO 13485 certified Quality Management System each lot of Norgen s Mycoplasma PCR Detection Kit including the Mycoplasma 2x PCR Master Mix Control 2x PCR Master Mix Isolation Control and Mycoplasma Positive Control are tested against predetermined specifications to ensure consistent product quality Product Use Limitations Norgen s Mycoplasma PCR Detection Kit is designed for research purposes only Product Warranty and Satisfaction Guarantee NORGEN BIOTEK CORPORATION guarantees the performance of all products in the manner described in our product manual The customer must determine the suitability of the product for its particular use
5. 7 bp Figure 1 A representative 1X TAE 1 5 agarose gel showing the amplification of Mycoplasma at different concentrations Lane 1 to 7 The size of the Mycoplasma target amplicon corresponds to 327 bp as represented by the provided DNA Marker M NTC Negative Control M 1 2 3 4 5 6 NTC Isolation Control lt PCR Control Figure 2 A representative 1X TAE 1 7 agarose gel showing the amplification of Isolation Control and PCR Control under different conditions using the Control 2X PCR Mastermix The size of the Isolation Control amplicon and PCR Control amplicon correspond to 499 bp and 150 bp respectively as represented by the provided DNA Marker M Lanes 1 to 5 showed detection of both Isolation Control and PCR Control suggesting that the DNA isolation as well as the PCR reaction was successful Lane 6 showed only the detection of PCR Control suggesting that while the PCR was successful the isolation failed to recover even the spiked in Isolation control NTC Negative Control Table 5 Interpretation of PCR Assay Results Input Type Target Control Reaction Interpretation reaction Mycoplasma IlsoC Band PCRC Band Target Band 499 bp 150 bp 327 bp a x x x Valid a x vali Sample x x x Positive Sample X X Negative Sample x Re test Sample Re test Sample X Negative Sample x x Positive Sample x x Positive Sample x Re test For results obtained that a
6. Fax 905 227 1061 BIOTEK si CORPORATION Email techsupport norgenbiotek com a 3430 Schmon Parkway K Thorold ON Canada L2V 4Y6 q Phone 866 667 4362 e 905 227 8848 Mycoplasma PCR Detection Kit Product Insert Product 33100 Pathogen Information Mycoplasma is a common and serious contaminants of cell cultures Mycoplasma is often not visible and does not respond to antibiotics and therefore it is a major issue that requires monitoring and early detection Up to 30 of cell cultures may be contaminated with Mycoplasmas the main contaminants being the species M orale A laidlawii M arginini and M hyorhinis These organisms produce a variety of effects on the infected cells in culture including changes in metabolism growth viability and morphology thereby altering the phenotypic characteristics of the host cells Therefore there is an absolute requirement for routine periodic assays to diagnose possible Mycoplasma contamination of all cell cultures particularly continuous or established cell lines Principle of the Test Norgen s MycoplasmaPCR Detection Kit constituents a ready to use system for the isolation and detection of Mycoplasma using end point PCR The kit first allows for the isolation of Mycoplasma DNA from the blood samples using spin column chromatography The Mycoplasma DNA is isolated free from inhibitors and can then be used as the template in a PCR reaction for Mycoplasma detection using the provided Mycoplasma
7. collection tube Note Ensure the entire lysate volume has passed through into the collection tube by inspecting the column If the entire lysate volume has not passed spin for an additional minute d Depending on your lysate volume repeat step 2c if necessary 3 Column Wash a Apply 500 uL of Wash Solution to the column and centrifuge for 1 minute Note Ensure the entire wash solution has passed through into the collection tube by inspecting the column If the entire wash volume has not passed spin for an additional minute b Discard the flowthrough and reassemble the column with its collection tube c Repeat step 3a to wash column a second time d Discard the flowthrough and reassemble the spin column with its collection tube e Spin the column for 2 minutes in order to thoroughly dry the resin Discard the collection tube 4 DNA Elution a Place the column into a fresh 1 7 mL Elution tube provided with the kit b Add 75 uL of Elution Buffer to the column c Centrifuge for 2 minutes at 2 000 x g 2 000 RPM followed by a 1 minute spin at 14 000 x g 14 000 RPM Note the volume eluted from the column If the entire volume has not been eluted spin the column at 14 000 x g 14 000 RPM for 1 additional minute 5 Storage of DNA The purified nucleic acids may be stored at 20 C for a few days It is recommended that samples be placed at 70 C for long term storage B Mycoplasma PCR Assay Preparation Notes B
8. ed Questions 1 How many samples should be included per PCR run e Norgen s Mycoplasma PCR Detection Kit is designed to test 24 samples For every 6 samples a non template control Nuclease Free Water and a Positive Control must be included It is preferable to pool and test 6 samples at a time If not the provided Positive Control is enough to run 3 samples at a time 2 How can interpret my results if neither the PCR control nor the Isolation Control soC amplifies e f neither the PCR control nor the Isolation Control soC amplifies the sample must be re tested If the positive control showed amplification then the problem occurred during the isolation where as if the Positive control did not amplify therefore the problem has occurred during the setup of the PCR assay reaction 3 How should it be interpreted if only the PCR control showed amplification but neither the Mycoplasma target nor the Isolation control amplified for a sample e This indicates a poor isolation The isolation procedure must be repeated 4 How should it be interpreted if only the Isolation Control soC was amplified in a sample e The sample tested can be considered as Mycoplasma negative 5 How should it be interpreted if the PCR control and the Mycoplasma target showed amplification in a sample e The sample tested can be considered positive It could happen when too much template was added to the reaction 6 How should it be interpreted if on
9. efore use suitable amounts of all PCR components should be completely thawed at room temperature vortexed and centrifuged briefly The amount of Mycoplasma 2X Detection PCR Master Mix and Control 2X PCR Master Mix provided is enough for up to 32 PCR reactions 24 sample PCR 4 positive control PCR and 4 no template control PCR For each sample one PCR reaction using the Mycoplasma 2X Detection PCR Mastermix and one PCR reaction using Control 2X PCR Mastermix should be set up in order to have a proper interpretation of the results For every PCR run one reaction containing Mycoplasma Positive Control and one reaction as no template control must be included for proper interpretation of results e The recommended minimum number of DNA samples tested per PCR run is 6 e Using a lower volume from the sample than recommended may affect the sensitivity of Mycoplasma Limit of Detection 1 Prepare the PCR reaction for sample detection Set 1 using Mycoplasma 2X Detection PCR Mastermix and the PCR reaction for control detection Set 2 using Control 2X PCR Mastermix as shown in Table 1 below The recommended amount of sample DNA to be used is 2 5 uL However a volume between 1 and 5 uL of sample DNA may be used as template Ensure that one Mycoplasma detection reaction and one control reaction is prepared for each DNA sample Adjust the final volume of the PCR reaction to 20 uL using the Nuclease Free Water provided Table 1 PCR Assay Pr
10. eparation PCR Components Volume Per PCR Reaction Sample DNA 2 5 uL Nuclease Free Water 7 5 uL Total Volume 20 uL 2 For each PCR set prepare one positive control PCR as shown in Table 2 below Table 2 PCR Positive Control Preparation PCR Components Volume Per PCR Reaction Total Volume 20 uL 3 For each PCR set prepare one no template control PCR as shown in Table 3 below Table 3 PCR Negative Control Preparation PCR Components Volume Per PCR Reaction Nuclease Free Water 10 uL Total Volume 20 uL C Mycoplasma PCR Assay Programming 1 Program the thermocylcer according to the program shown in Table 4 below 2 Run one step PCR Table 4 Mycoplasma Assay Program PCR Cycle Step Temperature Duration Cycle 1 Step 1 95 C 3 min Step 1 94 C 15 sec Cycle 2 40x Step 2 60 C 15 sec Step 3 72 C 30 sec Cycle 3 Step 1 72 C 5 min Cycle 4 Step 1 4 C D Mycoplasma PCR Assay Results Interpretation 1 For the analysis of the PCR data the entire 15 20 uL PCR Reaction should be loaded on a 1X TAE 1 7 Agarose DNA gel along with 10 uL of Norgen s DNA Marker provided 2 The PCR products should be resolved on the 1X TAE 1 5 Agarose gel at 150V for 30 minutes Gel running time will be vary depending on an electrophoresis apparatus 3 Sample results are provided below M 1 2 3 4 5 6 7 NTC M Mycoplasma gt 32
11. er equipment e g Scientific Industries Disruptor Genie Vortex for 5 minutes at maximum speed or optimize the condition for any commercially available bead beater equipment Note Foaming during the homogenization is common This foaming is due to detergents present in the Lysis Buffer and will not affect the protocol Incubate the Bead Tube with lysate at 65 C for 10 minutes Occasionally mix the lysate 2 or 3 times during incubation by inverting the tube Briefly spin the tube to remove liquid from the cap and transfer all of the lysate including cell debris to a DNase free microcentrifuge tube provided by the user by pipetting Ensure that the beads are not transferred during the pipetting Centrifuge the tube for 2 minute at 14000 x g 14 000 RPM Carefully transfer clean supernatant to a new DNase free microcentrifuge tube provided by the user without disturbing the pellet Note the volume Add an equal volume of 70 ethanol provided by the user to the lysate collected above 100 uL of ethanol is added to every 100 uL of lysate Vortex to mix Proceed to Step 2 Binding to Column 2 Binding DNA to Column a Assemble a spin column with one of the provided collection tubes b Add 10 uL of Isolation Control soC to the lysate mixture c Apply up to 600 uL of the lysate with ethanol onto the column and centrifuge for 1 minute at 14 000 x g 14 000 RPM Discard the flowthrough and reassemble the spin column with the
12. ly the Mycoplasma target and the PCR control were amplified in a sample e The sample tested can be considered as Mycoplasma positive 7 How should it be interpreted if only the Mycoplasma target was amplified in a sample e It is recommended that the isolation is repeated 8 How should it be interpreted if only the PCR control and the Isolation control showed amplification in a sample e The sample tested can be considered negative 9 What if forgot to do a dry spin after my third wash e Your first DNA elution will be contaminated with the Wash Solution This may dilute the DNA yield in your first elution and it may interfere with the PCR detection as ethanol is known to be a PCR inhibitor 10 What if forgot to add the Isolation Control soC during the isolation e It is recommended that the isolation is repeated 11 What if forgot to run the Control PCR for the sample and only ran the Detection PCR and obtained a positive result e The result can be considered positive However any negative result must be verified by running the associated control PCR to ensure that it is a true negative and not a false negative due to problems with the DNA isolation or the PCR reactions Related Products Product Fungi Yeast Genomic DNA Isolation Kit 27300 Bacterial Genomic DNA Isolation Kit 17900 Plant Fungi DNA Isolation Kit 26200 Technical Assistance NORGEN s Technical Service Department is staffed by expe
13. re not covered in Table 5 above please refer to the Troubleshooting Section E Mycoplasma PCR Assay Specificity and Sensitivity e The specificity of Norgen s Mycoplasma PCR Detection Kit is first and foremost ensured by the selection of the Mycoplasma specific primers as well as the selection of stringent reaction conditions The primers were checked for possible homologies to all in GenBank published sequences by sequence comparison analysis The specific delectability of all relevant strains has thus been ensured by a database alignment and by PCR amplification with the following bacteria commonly found in filed samples e Listeria Salmonella Streptococcus agalactiae Streptococcus dysglactiae Streptococcus uberis Staphylococcus aeurus E coli Mycoplasma F Linear Range e The linear range analytical measurement of Norgen s Mycoplasma PCR Detection Kit was determined by analysing a dilution series of a Mycoplasma quantification standards ranging from 1 x 10 cfu ul to 1 x 10 cfu pl e Each dilution has been tested in replicates n 4 using Norgen s Mycoplasma PCR Detection Kit on a 1X TAE 1 7 agarose gel e The linear range of Norgen s Mycoplasma PCR Detection Kit has been determined to cover concentrations from 24 fg to 2 4 ng e Under the conditions of the Norgen s Mycoplasma DNA Isolation procedure Norgen s Mycoplasma PCR Detection Kit covers a linear range from 24 copies to 2 4 x 10 copies Frequently Ask
14. rienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of NORGEN products If you have any questions or experience any difficulties regarding Norgen s Mycoplasma PCR Detection Kit or NORGEN products in general please do not hesitate to contact us NORGEN customers are a valuable source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at NORGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please contact our Technical Support Team between the hours of 8 30 and 5 30 Eastern Standard Time at 905 227 8848 or Toll Free at 1 866 667 4362 or call one of the NORGEN local distributors www norgenbiotek com or through email at techsupport norgenbiotek com 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 905 227 8848 Fax 905 227 1061 Toll Free in North America 1 866 667 4362 2013 Norgen Biotek Corp P133100 5
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