Manual innuprep Bacteria DNA Kit - IP-C96

Transcription

1 Manual innuprep Bacteria DNA Kit - IP-C96

2 Order No.: 845-IP IP reactions 480 reactions Publication No.: HB_IP-5596_e_ This documentation describes the state at the time of publishing. It needs not necessarily agree with future versions. Subject to change! Expression and further use permitted with indication of source. Copyright 201, Analytik Jena AG, AJ Innuscreen GmbH Manufacturer: AJ Innuscreen GmbH Robert-Rössle-Straße Berlin Made in Germany! Distribution/Publisher: Analytik Jena AG Konrad-Zuse-Straße Jena/Germany Phone +49 (0) / Fax +49 (0) / lifescience@analytik-jena.com

3 Contents Contents 1 Introduction Safety precautions Storage conditions Function testing and technical assistance Product use and warranty Kit components Recommended steps before starting Components not included in the kit Product specifications Principle of operation Lysis protocol for DNA Isolation from bacterial cell pellets after cultivation Preliminary steps of the InnuPure C Pre-filling of 96 Deep Well Plates and Elution Plate Preparing Stacker A of InnuPure C Automatic processing of the InnuPure C innuprep Bacteria DNA Kit IP-C96 Issue 04 /

4 Contents 2 Issue 04 / 2013 innuprep Bacteria DNA Kit IP-C96

5 Introduction 1 Introduction The innuprep Bacteria DNA Kit - IP-C96 has been designed for automated isolation of DNA from gram+ and gram- bacteria cell pellets using the InnuPure C96. The extraction procedure is based on a new-patented chemistry. The procedure combines an external lysis step of the bacteria cells using lysozyme with a subsequent proteolytic digestion step. After the lysis step the samples are transferred into the Reagent Plates of the kit, after prefilling all extraction reagents needed for the extraction process. The following extraction process runs automatically on the InnuPure C96. The extraction process is based on binding of the DNA on surface modified magnetic particles. After washing steps the nucleic acid is eluted from the magnetic particle with low salt buffer and is now ready to use. The extraction chemistry in combination with the InnuPure C96 protocol are optimized to get maximum of yield and quality. 2 Safety precautions All due care and attention should be exercised in handling the materials and reagents contained in the kit. Always wear gloves while handling these reagents and avoid any skin contact! In case of contact, flush eyes or skin with a large amount of water immediately. Use the kit once only! For skilled personal only! Don t eat or drink components of the kit! 3 Storage conditions The innuprep Bacteria DNA Kit - IP-C96 should be stored dry, at room temperature (14 25 C) and is stable for at least 6 months under these conditions. Before every use make sure that all components have room temperature. If there are any precipitates within the additional provided solutions solve these precipitates by careful warming. 4 Function testing and technical assistance The Analytik Jena AG guarantees the correct function of the kit for applications as described in the manual. The components of innuprep Bacteria DNA Kit - IP-C96 were tested by isolation of bacterial DNA from bacterial cell pellet, determination of DNA yield, Ratio A260/A280 and subsequent targetamplification. We reserve the right to change or modify our products to enhance their performance and design. If you have any questions or problems regarding any aspects of the innuprep Bacteria DNA Kit - IP-C96 or other Analytik Jena AG products, please do not hesitate to contact us. For technical support or further information in Germany please dial / For other countries please contact your local distributor. innuprep Bacteria DNA Kit IP-C96 Issue 04 /

6 Product use and warranty 5 Product use and warranty The user is responsible to validate the performance of the Analytik Jena AG kits for any particular use, since the performance characteristics of our kits have not been validated for any specific application. Analytik Jena AG kits may be used in clinical diagnostic laboratory systems after the laboratory has validated the complete diagnostic system as required by CLIA 88 regulations in the U.S. or equivalents in other countries. All products sold by the Analytik Jena AG are subjected to extensive quality control procedures and are warranted to perform as described when used correctly. Any problems should be reported immediately. Note For research use only! 4 Issue 04 / 2013 innuprep Bacteria DNA Kit IP-C96

7 Kit components 6 Kit components Important Store lyophilized Proteinase K at 4 C! Store the dissolved Proteinase K as described below! All other components are stored at room temperature. 96 extractions 480 extractions MAG Suspension 5,5 ml 3x 9 ml Lysis Solution CBV 30 ml 125 ml Proteinase K For 2 x 1.5 ml working solution Binding Solution SBS 50 ml 250 ml Washing Solution A (ready-to-use) Washing Solution B2 (ready-to-use) For 7 x 1.5 ml working solution 200 ml 3x 220 ml 250 ml 1000 ml RNase-free Water 125ml 3x 200 ml Elution Buffer 3x 25 ml 2x 200 ml 96 Deep Well Plate (2.2 ml) Filter Tips 1x 96 5x 96 Elution Plate 96 Plate (1.2 ml) 1 5 Manual 1 1 Initial steps Dissolve Proteinase K by addition of 1.5 ml of ddh 2 O, mix thoroughly and store as described below! Dissolve Proteinase K by addition of 1.5 ml of ddh 2 O, mix thoroughly and store as described below! Important note Store dissolved Proteinase K at 20 C. Repeated freezing and thawing will reduce the activity dramatically. Dividing of the Proteinase K into aliquots and storage at 20 C is recommended. innuprep Bacteria DNA Kit IP-C96 Issue 04 /

8 Recommended steps before starting 7 Recommended steps before starting Heat thermal mixer or water bath at 37 C and 50 C Ensure that the Proteinase K has been prepared according to the instruction ( "Kit components", p. 5) Centrifugation steps should be carried out at room temperature Avoid freezing and thawing of starting material 8 Components not included in the kit RNase A (100 mg/ml); optional TE buffer (1 mm EDTA; 10 mm Tris HCl, ph 7.5) Lysozym (10 mg/ml); dissolved in TE buffer 1.5 ml tubes 2.0 ml tubes; optional ddh 2 O 6 Issue 04 / 2013 innuprep Bacteria DNA Kit IP-C96

9 Product specifications 9 Product specifications 1. Starting material: Bacterial cell pellets after cultivation (max. 1 x 10 9 cells) 2. Time for isolation: Lysis: approx: min InnuPure C96 protocol standard: approx: 50 min 3. Typical yield: Not determined Depending on type and amount of the starting material innuprep Bacteria DNA Kit IP-C96 Issue 04 /

10 Principle of operation 10 Principle of operation Depending on the type and features of selected starting material, a given lysis process may have to be performed inside the system (internal process) or outside the system (external process) in manual mode. Refer to the user manual of a given extraction kit for a detailed description of lysis working steps/instructions for the corresponding type of starting material. Figure Step Explanation 1. Lysis (internal) 1. Lysis (external) For internal lysis, the necessary reagents are at first added to the samples in a sample plate and the sample plate is placed onto the left carriage position. This is followed by motion to transfer the plate into its working position below the pipetting head. In the majority of cases, the heating plate is then pressed against the well bottoms in order to process thermal pulpings. Continuous mixing throughout this working step will help the lysis process. For external lysis, a sample plate is equally placed onto the left carriage position. The automatic protocol routine begins with the workstep of binding. 2. Binding The procedure of binding nucleic acids to the magnetic particles begins with the addition of binding buffer. Once the binding buffer has been transferred, a homogeneous mixture of the various involved components is achieved by way of pipetting-on and pipetting-off steps performed in quick succession. 3. Collecting To collect the magnetic particles at the plate s well bottom, the heating magnet module below the plate moves into place at the plate. It moves exactly as far as necessary for the magnets to make direct contact with the well bottom through the heating plate. Any excessive binding buffer can thus be pipetted off and discarded. 4. Washing Any residual amount of magnetic particles at the well bottom can now be solved in a washing buffer. Repeated/multiple pipetting-on and pipetting-off cycles warrant proper mixing. Magnetic particles are subsequently collected again at the well bottom and any excessive amount is transferred back into the reagent plate that contains pre-filled washing buffer. This procedure is several times repeated using different 8 Issue 04 / 2013 innuprep Bacteria DNA Kit IP-C96

11 Principle of operation types of washing buffer. The number of required washing cycles, the required volume and the type of washing buffer are conditional on the type of selected starting material. 5. Transfer pipetting Depending on the particular protocol, the complete content of a plate is transferred into a new plate during and/ or on completion of a washing routine. This distinctly improves the quality of nucleic acids already washed. Any residual amount of lysis debris at the well walls will thus stay back in the latest washed plate and only those magnetic particles which have already been washed and are bound to nucleic acids will be subject to further transportation. 6. Tip washing In order to avoid any carry-over effects of residue potentially accumulating in the tips, the tips are subjected to rinsing in between certain washing steps. 7. Ethanol removal To remove residue of ethanol inside the wells, a drying workstep follows. This workstep consists in the heating plate of the heating magnet module being turned on and pressed against the bottom of the plate. 8. Elution Elution buffer is supplied. This is followed by heating of the mixture and homogenization by performing continuous pipetting-on and pipetting-off cycles. In the process nucleic acids get detached from the magnetic particles. As part of an elution workstep magnetic particles are again collected at the well bottom and the mixture is subsequently transferred to a new plate where the remaining magnetic particles are once more collected at the well bottom. 9. Eluate transfer Once all magnetic particle residues have gathered at the well bottom, the eluate is transferred into the elution plate. The software screen reports completion of the protocol sequence and the elution plate which is located in the left carriage position can now be retrieved from the system (after opening of the left door). Note Filling levels and filling colors are only shown for illustration and do not match the actual filling levels and colors of the kit reagents. innuprep Bacteria DNA Kit IP-C96 Issue 04 /

12 Lysis protocol for DNA Isolation from bacterial cell pellets after cultivation 11 Lysis protocol for DNA Isolation from bacterial cell pellets after cultivation Note The lysis of the starting material is a preliminary manual processing step. We recommend to start with the lysis and pre-filling the working/sample plate after the steps Pre-filling of 96 Deep Well Plates and Elution Plate 96, p.11. Note Max. amount of bacterial cell pellets after cultivation: 1 x 10 9 cells. 1. Add 200 µl TE buffer to the bacterial pellet and resuspend the pellet completely. Add 15 µl Lysozym (stock solution 10 mg/ml in TE buffer). Mix by pulsed vortexing for 5 sec. 2. Incubate at 37 C until the sample is lysed (approx min). Note: The lysis time depends very strong on type of starting material (e.g. gram+ or gram- bacteria). We recommend to use a shaking platform (thermomixer, water bath or another rocking platform) for a continuous shaking of the sample. Vortex the sample optionally during incubation. 3. Add 200 µl Lysis Solution CBV and 20 µl Proteinase K to the sample, mix vigorously by pulsed vortexing for 5 sec. Incubate at 50 C for 30 minutes. Note: We recommend to use a shaking platform (thermomixer, water bath or another rocking platform) for a continuous shaking of the sample. Vortex the sample optionally during incubation. No shaking will reduce the lysis efficiency! 4. Label one 96 Deep Well Plate with Working/Sample Plate and transfer the supernatant of samples into the wells. Note The lysed sample will be processed using the InnuPure C96. Please follow the instruction of the manual from point 13! 10 Issue 04 / 2013 innuprep Bacteria DNA Kit IP-C96

13 Preliminary steps of the InnuPure C96 12 Preliminary steps of the InnuPure C Pre-filling of 96 Deep Well Plates and Elution Plate 96 Note Before starting the sample preparation label the Deep Well Plates and pre-fill all needed buffers into the bottom of the wells of the Deep Well Plates and the Elution Plate 96 as described below! Reagent Plates (96 Deep Well) Binding Plate Buffer Add 400 µl Binding Solution SBS and 50 µl MAG Suspension to each well. Note! It is important to mix the MAG Suspension by vigorous shaking or vortexing before use (approx. 30 sec)! Washing Plate µl Washing Solution A Washing Plate µl Washing Solution A Washing Plate µl Washing Solution B2 Washing Plate µl Washing Solution B2 Washing Plate 5 Let all wells of the plate empty! Washing Plate µl RNase-free Water Washing Plate µl Elution Buffer Washing Plate 8 Let all wells of the plate empty! Working/Sample Plate After pre-fill all needed buffers into the wells of the Deep Well Plates as described below, follow the instructions Lysis protocol for DNA Isolation from bacterial cell pellets after cultivation (see page 10) to pre-fill the sample plate with your specific samples and lysis buffer. Note! It is important to pre-fill the working plate in the end of the pre-filling steps! Elution Plate 96 Plate (1,2 ml) Elution Plate Buffer Let all wells of the plate empty! innuprep Bacteria DNA Kit IP-C96 Issue 04 /

14 Preliminary steps of the InnuPure C Preparing Stacker A of InnuPure C96 Note Be sure that the pre-filled solutions are at the bottom of the 96 Deep Well Plates. Open the door of Stacker A (right Stacker) of the InnuPure C96 and load the pre-filled 96 Deep Well Plates as shown below: top bottom Stacker A Elution Plate Washing Plate 8 Washing Plate 7 Washing Plate 6 Washing Plate 5 Washing Plate 4 Washing Plate 3 Washing Plate 2 Washing Plate 1 Binding Plate! Important Note - Inserting 96 Deep Well Plates Always make sure that A1 faces toward the left back-end corner as you insert a Deep Well Plate! 12 Issue 04 / 2013 innuprep Bacteria DNA Kit IP-C96

15 Automatic processing of the InnuPure C96 13 Automatic processing of the InnuPure C96 1. Check for correct connection of power cord and USB cable. Turn InnuPure C96 into stand-by mode by transferring ON/OFF switch into position I. To switch the device on, keep the touch sensor depressed for 1.5 sec (approximately) the LED will change from red to green light. 2. Turn PC on and trigger the software Application Studio. 3. Start the extraction protocol: Use the radio button in the selection window (see page 44 in the device manual). Select the desired extraction protocol DNA_external_Lysis_C96_01 and press [START] Follow the instructions of the extraction protocol. Enter the recommended eluate volume of µl and press [OK]. Note: It is possible to adjust the volume values between 50 up to 500 µl. Find more detail information how to start an extraction protocol in InnuPure C96 Handbook 7.2 Software functions 44! innuprep Bacteria DNA Kit IP-C96 Issue 04 /

16 Automatic processing of the InnuPure C96 4. After completion of the protocol open the door of sample position. Note: The chosen protocol is performed by device and after the protocol is finished the message 'Purification process completed' is shown in the screen of the computer! 5. Remove the Elution Plate from the left carriage position of the InnuPure C96 with the final eluates containing the extracted DNA; close the Plate with a foil and store the DNA under proper conditions. 6. After finishing the extraction protocol, remove the used Deep Well Plates from Stacker B (left Stacker) and discard the Tip tray with used Filter Tips. Note After finishing the extraction protocol, the Elution Plate contains the extracted DNA. Store the DNA under adequate conditions. We recommend to store the extracted DNA at 20 C. 14 Issue 04 / 2013 innuprep Bacteria DNA Kit IP-C96

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18 Analytik Jena AG Life Science Konrad-Zuse-Strasse Jena / Germany Phone +49 (0) Fax +49 (0) lifescience@analytik-jena.com