MycoQuick mycoplasma detection kit User Manual
Contents
1. 650 968 2200 outside US For media Collect 1 1 5mL medium Centrifuge at 13000rpm for 5min Save pelletand wash w DPBS Add 100uL lysis buffer and lyse for 5min Boil for Smin and centrifuge Collect supernatant as PCR template Page 5 System Biosciences SBI User Manual B Experimental Procedure Cells cultured in the absence of antibiotics for several days can enhance the strength of PCR signal Media sample should be derived from cells that are at least 80 confluent Sample Preparation from Cell Lysate 1 Aspire medium from attached cells 2 Wash cells twice with DPBS 3 Add appropriate amount of lysis buffer 100 uL per well of 24 well plate Lyse at room temperature for 5 min and collect cell lysate to an eppendorf tube 4 Boil lysates at 95 C for 5 min 5 Centrifuge the lysates at 13000 rpm for 5 min and collect supernatant 6 Take 1 2 uL supernatant as template for PCR reaction Sample Preparation from Media 1 Collect 1 1 5 mL cell culture medium to an eppendorf tube 2 Centrifuge at 13000 rpm for 5 min 3 Discard supernatant and wash the pellet once with DPBS 4 Add 100 ut lysis buffer to the pellet pippet up and down and lyse at room temperature for 5 min 5 Boil lysates at 95 C for 5 min 6 Centrifuge the lysates at 13000 rpm for 5 min and collect supernatant 7 Take 1 2 uL supernatant as template for PCR reaction PCR Reaction MycoQuick 96 well mycoplasma detection plate has b2. 3 Electrophorese at the conditions recommended by the gel box manufacturer 4 Visualize the bands with ethidium bromide C Expected Results To determine whether the sample is contaminated with mycoplasma insure that both positive control and negative control give expected results PCR template PCR product s Interpretation Positivecdntrol Th 2 ang 2052p Expected control result One No band Failed PCR reaction Negative control No band Expected result One Two bands Contaminated reagents 280 bp and 205 bp Mycoplasma bands contamination Heavy mycoplasma 280 bp band only contamination Sample No mycoplasma 205 bp band only Satna No band Inhibited PCR reaction If the cell culture is contaminated with mycoplasma a 280 bp band will be observed on the gel In addition a 205 bp band should be amplified from the UCE of the genomic DNA of the hosting cells If the cell culture is heavily infected with mycoplasma the amplification of the 280 bp band may result in diminished amplification of the 205 bp internal control Media samples give a weaker UCE internal control signal than the cell lysate samples Page 8 ver 1 030811 www systembio com MycoQuick Mycoplasma Detection Kit An example of experimental result is shown below L Cell lysate M Media sample L1 Mi L2 M2 L3 M3 lt 280 bp lt 205bp Both cell lysate and medium from sample 1 give positive result for m
3. Hyorhinis M Arginini M Orale M Fermentans A laidlawii which represent 98 of tissue culture infections It can also detect Mycoplasma Acholeplasma Ureaplasma Spiroplasma from other genera Page 2 ver 1 030811 www systembio com MycoQuick Mycoplasma Detection Kit A positive control is included to control the successful amplification of PCR reaction and confirm the size of the PCR products in the testing samples The mycoplasma primers will generate a 280 bp product The UCE primers will generate a 205 bp band The UCE primers confirm the absence of PCR inhibitors in all PCR samples The UCE primers have been tested to detect an ultra conserved element in human mouse and rat genome The amplification of UCE indicates a successful sample preparation and subsequent PCR amplification which excludes false negative results Both mycoplasma primers and UCE primers are coated at the bottom of the each well of 96 well plate Two amplifications can be achieved simultaneously in one PCR reaction The two PCR products can be easily separated from each other on a 2 5 agarose gel The primers have been adapted for using a variety of PCR enzymes such as Taq Pfu Phusion and SYBR Green mix D Precautionary Notes 1 To prevent contamination of mycoplasma DNA during experimental procedure always wear gloves during sample preparation and PCR reaction setup 2 To avoid false positives water used in PCR reactions can be UV irradiate
4. S Page 11
5. SSBI System Biosciences MycoQuick Mycoplasma Detection Kit User Manual Store Detection plate at 4 C Store lysis buffer at 20 C A limited use label license covers this product By use of this product you accept the terms and conditions outlined in the Licensing and Warranty Statement ver 1 030811 contained in this user manual MycoQuick Mycoplasma Detection Kit Contents l Introduction and Background Additional Materials and Instruments Needed onmog0ow gt Il Protocols icin ee Re as w gt C Expected ReSUuIts cccscceesceceeeeeeeeeeeeeaeeeeeeesnnees IIL REPCPENCE wisi ciliate neta vanaeiel a ie IV Technical Support cccceeceeeseeeeeeeeeeeeeeesaeeseneeeeees V Licensing and Warranty s es Quick Reference See sample results on Page 9 888 266 5066 Toll Free 650 968 2200 outside US Mycoplasma Contamination ccceeseeeeeeeeeees Mycoplasma Detection Methods cee MycoQuick Mycoplasma Detection Kit Precautionary Notes ccccceeeeseeeeeeeseeeeeeeneeeeeenas Kit Components cceceeeeeeeeeeeceeeeeceaeeeeaeeseeeeeeaees SS Storage is oaaae aa aeaaea eee Howechariinisisnni n a Experimental Procedure asssssssssssrresrrrresrrreerennes Page 1 System Biosciences SBI User Manual Introduction and Background A Mycoplasma Contamination Mycoplasmas are small round or filamentous prokaryoti
6. c organisms which are a frequent contaminant of cell cultures It has been estimated that 15 35 of cells cultures are contaminated with mycoplasma The contamination can modify many aspects of cell physiology and result in unreliable experimental results Due to their small size and deformability mycoplasma can pass or be forced through 0 22 um filters The lack of a cell wall makes mycoplasma unresponsive to common antibiotics It is essential that all cell stocks and new cultures entering a facility are tested for the presence of mycoplasma and all cell cultures should be tested routinely e g once every 2 3 months in order to maintain a mycoplasma free environment B Mycoplasma Detection Methods Many methods are available for detection of mycoplasma including isolation in broth agar culture direct 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 Ref C MycoQuick Mycoplasma Detection Kit The SBI MycoQuick mycoplasma detection kit is capable of detecting mycoplasma infection in cell cultures in less than three hours It can detect mycoplasma from both cell lysates and cell culture media The sensitivity is up to 10 20 copies of target DNA which translates to less than 10 mycoplasma The MycoQuick mycoplasma detection kit detects the top five common species of mycoplasma M
7. d 3 To avoid cross contamination between samples aerosol resistant pipet tips should be used throughout the protocol 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual E Kit Components 1 2 3 MycoQuick 96 well plate with mycoplasma primers and UCE primers coated at the bottom of each well The 12 wells of the last lane also contain positive control DNA templates for mycoplasma and UCE The plate can be cut into strips according to the number of the samples to be tested LAANA AAAA X os lt gt KAJ A N a a a AAAA J AA INA NSA NA NA NYY Positive Control O A IN 8 cap strips x12 Cell lysis buffer 10 ml F Additional Materials and Instruments Needed 1 Table top centrifuge 2 PCR enzyme and buffer 3 PCR machine qPCR machine 4 Materials and apparatus for DNA electrophoresis G Storage Store 96 well MycoQuick mycoplasma detection plate at 4 C until the expiration date Store cell lysis buffer at 4 C or 20 C until the expiration date Page 4 ver 1 030811 www systembio com MycoQuick Mycoplasma Detection Kit ll Protocols A Flowchart Overview For cell lysate Aspire medium and wash twice w DPBS Add 100pL lysis buffer 1 well in 24 well plate D Lyse for Smin and collectcell lysates Boil for 5min and centrifuge Collect supernatant as PCR template 888 266 5066 Toll Free
8. een tested for various PCR enzymes including Taq Pfu Phusion and SYBR Green mix PCR setup may vary according to the enzyme used A typical setup with Taq is shown below A final volume of 25 uL is recommended for each reaction Page 6 ver 1 030811 www systembio com MycoQuick Mycoplasma Detection Kit Component Sample reaction control reaction control reaction P 25 uL 25 uL 25 uL 10xBuffer 2 5 2 5 25 dNTP mix j 10 mM Sample 1 Taq polymerase 0 2 0 2 0 2 hzo 20 3 21 3 21 3 The general order for setting up PCR is to make the PCR master mix first add the appropriate amount of mix e g 24 uL to each well of 96 well plate and then add 1 uL of cell lysate medium sample to each well Close the wells tightly with the cap strips provided and put them into PCR machine A typical PCR program with Taq is shown below Cycle s Temperature Time 1 95 C 30s 95 C 30s 40 56 C 30s 68 C 30s 1 68 C 5 min Electrophoresis of PCR Products 888 266 5066 Toll Free For optimal separation between the mycoplasma band and the UCE band we recommend 2 5 agarose gel for electrophoresis 1 Mix the final products of each PCR reaction with gel electrophoresis loading buffer 2 Load each sample together with DNA size marker into individual well of 2 5 agarose gel 650 968 2200 outside US Page 7 System Biosciences SBI User Manual
9. is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use Page 10 ver 1 030811 www systembio com MycoQuick Mycoplasma Detection Kit Limited Warranty SBI warrants that the Product meets the specifications described in the accompanying Product Analysis Certificate If it is proven to the satisfaction of SBI that the Product fails to meet these specifications SBI will replace the Product or provide the purchaser with a refund This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to SBI within 30 days of receipt of the Product SBI s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price SBI s liability does not extend to any damages arising from use or improper use of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty SBI does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose SBI is committed to providing our customers with high quality products If you should have any questions or concerns about any SBI products please contact us at 888 266 5066 2011 System Biosciences SBI All Rights Reserved 888 266 5066 Toll Free 650 968 2200 outside U
10. ycoplasma with both 280 bp and 205 bp bands or 280 bp band only Sample 2 and 3 give negative result with 205 bp band only lll References Detection of Mycoplasma in cell cultures Young L Sung J Stacey G Masters JR Nat Protoc 2010 5 5 929 34 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual IV Technical Support For more information about SBI products and to download manuals in PDF format please visit our web site http www systembio com For additional information or technical assistance please call or email us at Phone 650 968 2200 888 266 5066 Toll Free Fax 650 968 2277 E mail General Information info systembio com Technical Support tech systembio com Ordering Information orders systembio com System Biosciences SBI 265 North Whisman Road Mountain View CA 94043 V Licensing and Warranty Use of the MycoQuick mycoplasma detection kit ie the Product is subject to the following terms and conditions If the terms and conditions are not acceptable return all components of the Product to System Biosciences SBI within 7 calendar days Purchase and use of any part of the Product constitutes acceptance of the above terms The purchaser of the Product is granted a limited license to use the Product under the following terms and conditions The Product shall be used by the purchaser for internal research purposes only The Product
Download Pdf Manuals
Related Search