SARTORIUS SMB95 Microsart Calibration Reagent
For Mollicutes and other bacteria species
- Prod. No. SMB95-2021 Mycoplasma arginini
- Prod. No. SMB95-2022 Mycoplasma orale
- Prod. No. SMB95-2023 Mycoplasma gallisepticum
- Prod. No. SMB95-2024 Mycoplasma pneumoniae
- Prod. No. SMB95-2025 Mycoplasma synoviae
- Prod. No. SMB95-2026 Mycoplasma fermentans
- Prod. No. SMB95-2027 Mycoplasma hyorhinis
- Prod. No. SMB95-2028 Acholeplasma laidlawii
- Prod. No. SMB95-2029 Spiroplasma citri
- Prod. No. SMB95-2030 Bacillus subtilis
- Prod. No. SMB95-2031 Pseudomonas aeruginosa
- Prod. No. SMB95-2032 Kocuria rhizophila
- Prod. No. SMB95-2033 Clostridium sporogenes
- Prod. No. SMB95-2034 Bacteroides vulgatus
- Prod. No. SMB95-2035 Staphylococcus aureus
- Prod. No. SMB95-2036 Mycoplasma salivarium
For use in research and quality control
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Intended Use
Microsart® Calibration Reagent is titrated genomic DNA and can be used as amplification and sensitivity control for endpoint PCR (gel-based evaluation). If quantitative PCR is required, titrated genomic DNA can be serially diluted and used as PCR templates to create standard curves. The software will generate a standard curve from the known DNA dilutions and use it to determine the DNA concentration of the unknown samples.
Explanation of the Product
This product provides isolated genomic DNA of a specific species. An early passage strain of the specific microorganism is inoculated in a suitable culture medium and harvested at the end of the logarithmic growth phase by repeated washing and centrifugation. Following extraction, the DNA concentration is quantified photometrically (OD260/280) and with a double-stranded DNA (dsDNA)-specific ultrasensitive fluorescent dye, calibrated with weight reference standards and controlled by qPCR (compared to exactly quantified calibrator plasmids). The DNA concentration was adjusted with a TE80 buffer.
The following species are available:
- Mycoplasma arginini ATCC 23838; NCTC 10129
- Mycoplasma orale ATCC 23714; NCTC 10112; DSM 25590
- Mycoplasma gallisepticum ATCC 19610; NCTC 10115; DSM 19817
- Mycoplasma pneumoniae ATCC 15531; NCTC 10119; DSM 22911
- Mycoplasma synoviae ATCC 25204; NCTC 10124; DSM 21430
- Mycoplasma fermentans ATCC 19989; NCTC 10117
- Mycoplasma hyorhinis ATCC 17981; NCTC 10130; DSM 25591
- Acholeplasma laidlawii ATCC 23206; NCTC 10116; DSM 23060
- Spiroplasma citri ATCC 27556; NCTC 10164; DSM 21846
- Bacillus subtilis ATCC 6633; NCTC 10400; DSM 347
- Pseudomonas aeruginosa ATCC 9027; DSM 1128
- Kocuria rhizophila ATCC 9341; DSM 348
- Clostridium sporogenes ATCC 19404; NCTC 532; DSM 1664
- Bacteroides vulgatus ATCC 8482; DSM 1447
- Staphylococcus aureus ATCC 6538; NCTC 10788; DSM 799
Principle
Each vial contains 1×108 copies of the complete bacterial genome. The genomic DNA material can be used directly for PCR and cannot be used for culture methods.Please note: For process validation, alternatively the corresponding Microsart® Validation Standards can be used (see Related Products).
Notes on the Procedure
- For in vitro use in research and quality control. This kit should be used only by trained staff.
- This kit does not contain hazardous substances and may be disposed of according to local regulations.
- This leaflet must be fully understood in order to successfully use the Microsart®Calibration Reagent. The reagents supplied should not be mixed with reagents from different lots and used as an integral unit. The reagents of the kit should not be used beyond their shelf life.
- Any deviation from the described method can affect the results.
- For each test setup, at least one negative control should be added. Participation in external quality control programs, such as those offered by Minerva Biolabs GmbH (www.minerva-biolabs.com), is recommended.
Reagents
Each kit contains 1 vial of bacterial DNA (1×108 genome copies) and 3 vials of buffer for the preparation of dilutions and negative controls. Samples are lyophilized for product stability reasons. The material has been inactivated prior to lyophilization and can be considered as non-infectious. The expiry date of the unopened product is specified on the package label. The kit components are stored at +2 to +8 °C upon arrival, at low humidity conditions. Lyophilized components must be stored ≤ -18 °C after rehydration.
The lot specific Certificates of Analysis can be downloaded from the MySartorius portal (https://my.sartorius.com).
Needed but not included
Microsart® Calibration Reagent contains all necessary positive and negative material. General industrial supplies and reagents, usually available in PCR laboratories are not included:
Consumables
- Laboratory gloves
- DNA-free pipette filter tips (Biosphere® filter tips from Sarstedt are recommended: 0.5-20 μl, Prod. No. 70.1116.210; 2-100 μl, Prod. No. 70.760.212; 20-300 μl, Prod. No. 70.765.210; 100-1000 μl. Prod. No. 70.762.211)
- 1.5 ml reaction tubes, DNA- and RNA-free
Equipment
Microcentrifuge for 1.5 ml reaction tubes
Vortex
Pipettes (Sartorius)
- mechanical
- 0.5 – 10 μl Sartorius Prod. No. LH-729020
- 10 – 100 μl Sartorius Prod. No. LH-729050
- 100 – 1000 μl Sartorius Prod. No. LH-729070
- or electrical
- 0.2 – 10 μl Sartorius Prod. No. 735021
- 10 – 300 μl Sartorius Prod. No. 735061
- 50 – 1000 μl Sartorius Prod. No. 735081
Rack for 1.5 ml tubes
For PCR analysis, a bacteria- or mycoplasma-specific DNA detection system is required.
- For bacterial DNA, we recommend the Microsart® ATMP Bacteria kit (Sartorius Prod. No. SMB95-1008), Microsart® ATMP Sterile Release kit (Sartorius Prod. No. SMB95- 1007), or the Microsart® Research Bacteria kit (Sartorius Prod. No. SMB95-1009).
- For detection of mycoplasma DNA, we recommend the Microsart® ATMP Mycoplasma (Sartorius Prod. No. SMB95-1003/1004), Microsart® AMP Mycoplasma (Sartorius Prod. No. SMB95-1001/1002), or Microsart® Research Mycoplasma (Sartorius Prod. No. SMB95-1005/1006).
Test Procedure
Rehydration of the reagents
All reagents must be equilibrated to room temperature prior to use.
- Centrifuge the vial containing the Calibration Reagent (green cap) briefly to collect the lyophilized material at the bottom.
- Add 100 μl Buffer (white cap) to obtain a concentration of 1×106 genomes/ μl.
- Incubate 5 min at room temperature.
- Vortex for 10 sec and spin for 5 sec with the “pulse” option or at 5000 × g.
- Aliquot the vial content in DNA-free tubes and freeze at ≤−18 °C or directly proceed to step 7.2.
Preparation of the dilutions
- Thaw the resuspended Calibration Reagent if applicable.
- Label six 1.5 ml reaction tubes as in the figure below and fill each with 90 μl of Buffer (white cap).
- Vortex Calibration Reagent briefly (at least 5 sec) at medium speed.
- Add 10 μl of the Calibration Reagent to reaction tube no. 1, close the tube and vortex briefly at medium speed.
- Add 10 μl of the content of reaction tube no. 1 to reaction tube no. 2.
- Close the tube and vortex briefly at medium speed.
- Proceed with the following reaction tubes of the dilution series in the same way.
PCR
Please follow the instructions given in the manual of the specific PCR kit. The volume used as a template for PCR defines the number of genome copies per reaction:
| Reaction tube | 2 µl sample volume | 10 µl sample volume | 50 µl sample volume |
| 1. | 2×105 genome copies | 1×106 genome copies | 5×106 genome copies |
| 2. | 2×104 genome copies | 1×105 genome copies | 5×105 genome copies |
| 3. | 2×103 genome copies | 1×104 genome copies | 5×104 genome copies |
| 4. | 200 genome copies | 1000 genome copies | 5000 genome copies |
| 5. | 20 genome copies | 100 genome copies | 500 genome copies |
| 6. | 2 genome copies | 10 genome copies | 50 genome copies |
Evaluation
In qPCR, the Ct values should decrease linearly with increasing DNA concentrations per reaction when a suitable PCR assay is used. The software of the qPCR device builds a standard curve and calculates the slope using the DNA concentrations provided by the user and the obtained Ct values. Also, the unknown sample DNA concentrations are automatically determined by the interpolation of the Ct values obtained for these samples on the standard curve. The following results represent an example of such evaluation and were generated using the Mx3005P™ instrument.
- Fig. 1: Amplification curves of a dilution series from 1×107 to 10 genome copies/reaction (GE/PCR).
- Fig. 2: Standard curve generated with the Mx3005P™ instrument using second derivative maximum method and the data from Fig. 1
Detection Kits for qPCR
Limited Product Warranty
This warranty limits our liability for the replacement of this product. No warranties of any kind, express or implied, including, without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided. Minerva Biolabs shall have no liability for any direct, indirect, consequential, or incidental damages arising out of the use, the results of use, or the inability to use this product.
Trademarks
Microsart is a registered trademark of Sartorius. Mx3005P is a trademark of Agilent Technologies. Last technical revision:2020-01-10
Sartorius Lab Instruments GmbH & Co. KG
- Otto-Brenner-Str. 20
- 37079 Goettingen, Germany
- Phone +49 551 308 0
- Fax +49 551 308 3289
- www.sartorius.com
Copyright by Sartorius, Goettingen, Germany. All rights reserved. No part of this publication may be reprinted or translated in any form or by any means without the prior written permission of Sartorius. The status of the information, specifications and illustrations in this manual is indicated by the date given below. Sartorius reserves the right to make changes to the technology, features, specifications and design of the equipment without notice. Status: September 2024, Sartorius, Goettingen, German.y Printed in Germany on paper that has been bleached without any use of chlorine. | W
Publication No.: SM-6112-e240901
Order No.: 85037-546-77
Ver. 09 | 2024
FAQ
- Q: Can this product be used for clinical diagnostics?
- A: No, this product is intended for research and quality control purposes only.
- Q: How should I store the product?
- A: Store the product at the specified conditions mentioned in the manual to maintain its integrity.
Documents / Resources
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SARTORIUS SMB95 Microsart Calibration Reagent [pdf] Instruction Manual SMB95-2021, SMB95-2022, SMB95-2023, SMB95-2024, SMB95-2025, SMB95-2026, SMB95-2027, SMB95-2028, SMB95-2029, SMB95-2030, SMB95-2031, SMB95-2032, SMB95-2033, SMB95-2034, SMB95-2035, SMB95-2036, SMB95 Microsart Calibration Reagent, Microsart Calibration Reagent, Calibration Reagent |