MycoGuard - GeneCopoeia
Contents
1. GeneCopoeia i Expressway to Discovery MycoGuard Mycoplasma PCR Detection Kit For quick detection of mycoplasma contamination Cat No MPD T 050 50 PCR reactions User Manual GeneCopoeia Inc 9620 Medical Center Drive 101 Rockville MD 20850 USA 301 762 0888 866 360 9531 inquiry genecopoeia com www genecopoeia com 2013 GeneCopoeia Inc MycoGuard Mycoplasma PCR Detection Kit Manual USER MANUAL MycoGuard Mycoplasma PCR Detection Kit l Description II Contents and Storage Ill Sample Preparation IV PCR Reaction V Limited Use License and Warranty I Description The MycoGuard mycoplasma PCR detection kit is a quick simple and sensitive PCR based mycoplasma detection kit The kit contains an optimized master mix ultrapure water and a positive control It specifically detects mycoplasma in cell cultures or cell culture derived products The primers in the MycoGuard master mix are highly specific to the conserved rDNA region in the mycoplasma genomes and can detect all well known mycoplasma genera including the commonly encountered ones in cell cultures such as M arginini M arthritidis M bovis M fermentans M genitalium M hominis M hyorhinis M neurolyticum M orale M pirum M pneumoniae M pulmonis M salivarium and U urealyticum Mycoplasma positive samples can be easily recognized by a distinct PCR product ranging in size from 150 to 400 bp Key advantages Simple2. Use 0 1 10 pl of the boiled sample for PCR Note It is not necessary to remove cells or cell debris from the culture medium The boiled samples can be stored at 4 C for 1 2 months Option 3 Isolate mycoplasma DNA from cultured cells The following procedure is recommended for cell lines whose growth is inhibited by mycoplasma 1 Transfer 1 10x10 of cells to a 1 5 ml tube Centrifuge the tubes for 1 minute at 10 000 g to pellet cells Carefully aspirate the supernatant 2 Suspend cell pellet with 1 ml of PBS Centrifuge the tubes for 1 minute at 10 000x g at room temperature Carefully aspirate the supernatant 3 Wash the pellet one more time with PBS Discard supernatant 4 Suspend cell pellet with 100 ul of molecular biology grade water Incubate the tube for 5 10 minutes at 95 C with the cap tightly closed Centrifuge the tube for 1 minute at 20 000xg to pellet denatured proteins MycoGuard Mycoplasma PCR Detection Kit Manual 6 Use 0 1 10 pl of the boiled sample for PCR Note The boiled samples can be stored at 4 C for 1 2 months IV Set up PCR reaction 1 Thaw the MycoGuard Master Mix on ice Tap the tube gently to mix the thawed solution Spin the tube briefly 2 The volume of each PCR reaction is 20 ul Both negative and positive control reactions should be included Set up PCR reaction tubes on ice Use 5 0 ul 4xMaster Mix per one reaction Negative control Positive control Test Sample Ultrapure w
3. ater u 4xMaster Mix pl Mycoplasma W Tx Final vol ul 3 Cap the tubes after adding all of the components Tap the tubes to mix the contents Spin the tubes briefly to bring the contents to the bottom Proceed immediately to the following PCR reaction 94 C 1 min 4 94 C 15 sec 4 50 C 20 sec 40 cycles L 72 C 20 sec L 72 C 2 min 4 Analyze the PCR products by 2 agarose gel electrophoresis Non treated culture medium Boiled culture medium 7 8 9 10 Figure 1 Example of mycoplasma PCR M 100 bp DNA ladder C Negative control 1 5 non treated cell culture medium 0 03 0 1 0 3 1 0 and 3 0 ul respectively 6 10 boiled cell culture medium 0 03 0 1 0 3 1 0 and 3 0 ul respectively Note Serum proteins and other components in the cell culture medium can inhibit the PCR reaction MycoGuard Mycoplasma PCR Detection Kit Manual V Limited Use License and Warranty Limited Use License Following terms and conditions apply to use of all MycoGuard Mycoplasma PCR Detection Kit the Product If the terms and conditions are not acceptable the Product in its entirety must be returned to GeneCopoeia within 5 calendar days A limited End User license is granted to the purchaser of the Product The Product shall be used by the purchaser for internal research purposes only The Product is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use The Product must not
4. be resold repackaged or modified for resale or used to manufacture commercial products without prior written consent from GeneCopoeia This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic research Use of any part of the Product constitutes acceptance of the above terms Limited Warranty GeneCopoeia warrants that the Product meets the specifications described in the accompanying Product Datasheet If it is proven to the satisfaction of GeneCopoeia that the Product fails to meet these specifications GeneCopoeia will replace the Product In the event a replacement cannot be provided GeneCopoeia will 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 GeneCopoeia within 30 days of receipt of the Product GeneCopoeia s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price GeneCopoeia 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 GeneCopoeia does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose GeneCopoeia is committed to providing our customers
5. culture medium to a 1 5 ml tube Incubate the tightly capped tube for 5 10 minutes at 95 C Dilute the boiled samples 20 fold with molecular biology grade water Centrifuge the tube for 1 minute at 20 000xg to pellet denatured proteins Then use 2 6 ul of diluted culture medium equivalent to 0 1 0 3 ul of original culture medium for PCR Note For suspension cells it is not necessary to remove cells from the culture medium The volume of culture medium should not exceed 1 0 ul per one 20 ul PCR reaction Serum proteins and other components in the cell culture medium can inhibit the PCR reaction The boiled samples can be stored at 4 C for 1 2 months Option 2 Enrich mycoplasma DNA from culture medium To help detect low abundant mycoplasma in the culture it is necessary to enrich mycoplasma using the following procedure before PCR analysis 1 Transfer 1 ml of culture medium to a 1 5 ml tube Centrifuge the tubes for 5 minutes at 20 000xg at room temperature Carefully aspirate the supernatant 2 Suspend the pellet with 1 ml of PBS Centrifuge the tubes for 5 minutes at 20 000xg at room temperature Carefully aspirate the supernatant 3 Wash the pellet 2 more times with PBS Carefully aspirate the supernatant 4 Suspend the pellet with 100 ul of molecular biology grade water Incubate the tube for 5 10 minute at 95 C with the cap tightly closed Centrifuge the tube for 1 minute at 20 000xg to pellet denatured proteins 6
6. e PCR reaction is ready to go No need to pre treat your cell culture medium Fast Get the result in as short as 2 hours Sensitive e Require as little as 0 03 ul of cell culture medium ll Contents and Storage Content Quantity Storage and shipping conditions 4x MycoGuard master 250 ul Stored at 20 C Avoid freeze thaw cycles Divide it into as smaller aliquots if necessary Shipped on Ice pack Ultrapure water 1 0 ml Stored at room temperature Shipped on Ice pack Stored at 20 C Positive control 20 ul ee Shipped on Ice pack Additional materials equipments required but not supplied Thermo cycler 100 bp DNA ladder Agarose gel Phosphate buffered saline PBS cell culture grade 1 5 ml centrifuge tubes with cap 0 2 ml thin wall PCR reaction tubes Horizontal electrophoresis apparatus and power supply MycoGuard Mycoplasma PCR Detection Kit Manual lll Sample preparation IMPORTANT NOTES Prior to PCR detection the cells should have been grown continuously for over 2 weeks in the absence of any antibiotics The cells should be over 80 confluent and the culture medium should be at least 2 days old at the time of sampling Option 1 Fast and simple procedure 1 Remove 2 5 ul of culture medium and dilute 20 fold with molecular biology grade water Then use 2 6 ul of diluted culture medium equivalent to 0 1 0 3 ul of original culture medium for PCR 2 Alternatively transfer 50 ul of
7. with high quality products If you should have any questions or concerns about any GeneCopoeia products please contact us at 301 762 0888 2013 GeneCopoeia Inc GeneCopoeia Inc 9620 Medical Center Drive Rockville MD 20850 Tel 301 762 0888 Fax 301 762 3888 Email inquiry genecopoeia com Web www genecopoeia com GeneCopoeia Products are for Research Use Only Copyright 2013 GeneCopoeia Inc Trademarks GeneCopoeia OmicsLink All in One EndoFectin GeneCopoeia Inc SYBR Molecular Probes iQ 5 Bio Rad ROX Invitrogen MPD 021913 4
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