Manual innuprep Plant DNA I Kit - IPC16

Transcription

1 Manual

2 Order No.: 845-IPS IPS IPP IPP IPP IPS = Kit contains prefilled reagent strips for processing individual samples IPP = Kit contains prefilled reagent plates for running 8 samples in parallel Note: Prefilled reagent strips and reagent plates can be used in parallel in the InnuPure C16. Publication No.: HB_IPX -1516_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 2014, Analytik Jena AG, AJ Innuscreen GmbH Manufacturer: AJ Innuscreen GmbH Robert-Rössle-Straße Berlin Made in Germany! 16 reactions 96 reactions 16 reactions 96 reactions 480 reactions Distribution/Publisher: Analytik Jena AG Konrad-Zuse-Straße Jena/Germany Phone +49 (0) / Fax +49 (0) / lifescience@analytik-jena.com ISO

3 Contents Contents 1 Introduction Intended use Notes on the use of this manual 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 Homogenization and lysis of plant samples Protocols: DNA isolation from plant material Protocol 1: gdna from plant using Lysis Solution SLS Protocol 2: gdna from plant using Lysis Solution OPT Protocol 3: gdna from plant using Lysis Solution CBV Preliminary steps of the InnuPure C Sample tray Preparing sample tray Sample Automatic processing of the InnuPure C Related Products innuprep Plant DNA I Kit IPC16 Issue 01 /

4 Contents 2 Issue 01 / 2014 innuprep Plant DNA I Kit IPC16

5 Introduction 1 Introduction 1.1 Intended use The innuprep Plant DNA I Kit - IPC16 has been designed for automated isolation of DNA from plant sample using the InnuPure C16. The extraction procedure is based on a new-patented chemistry. The protocol is based on using mortar and pestle and liquid nitrogen for homogenization. The homogenization is followed by an external lysis/cleaning step. For lysis of plant material the kit contains three different Lysis Solutions. These are Lysis Solution SLS, Lysis Solution OPT and Lysis Solution CBV. All three Lysis Solutions contain a mixture of chaotropic and anti-chaotropic salts (DC-Technology), detergents and other additives. Dirty lysates will be cleared by a centrifugation based precipitation and/or filtration using a Prefilter (lavender) to remove polysaccharides, contaminations and residual cellular debris. The samples are then transferred into the Reagent Strips or Reagent Plate of the kit, which is already prefilled with all extraction reagents needed for the extraction process. The following extraction process runs automatically on the InnuPure C16. 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 particles with low salt buffer and is now ready to use. The extraction chemistry in combination with the InnuPure C16 protocol are optimized to get maximum of yield and quality. The innuprep Plant DNA I Kit IPC16 allows processing of up to mg (dry weight) or mg (wet weight) starting material. The kit has been tested for isolation of gdna from leafs, fruits, woods, needles as well as seeds. The starting material can be fresh or frozen. Depending on the individual sample typical yields are in the range from 3 25 µg DNA (more yield are also possible). For any photometric and blank measurements an additional reaction tube containing separate Elution Buffer is included in the scope of delivery. Note For difficult starting materials, such as wood samples or samples with a high proportion of phenol, we recommend the innuprep Plant DNA II Kit - IPC16! Consult instruction for use This package insert must be read carefully prior to use. Package insert instructions must be followed accordingly. Reliability of results cannot be guaranteed if there are any deviations from the instructions in this package insert. innuprep Plant DNA I Kit IPC16 Issue 01 /

6 Introduction 1.2 Notes on the use of this manual For easy reference and orientation, the manual uses the following warning and information symbols as well as the shown methodology: REF Catalogue number N Content Contains sufficient reagents for <N> reactions Storage conditions Store at room temperature or shown conditions respectively Consult instructions for use This information must be observed to avoid improper use of the kit and the kit components. Used by Expiry date. Lot number The number of the kit charge Manufactured by Contact information of manufacturer For single use only Do not use components for a second time. Note / Attention Observe the notes marked in this way to ensure correct function of the device and to avoid operating errors for obtaining correct results. The following systematic approach is introduced in the manual: The chapters and figures are numbered consecutively. A cross reference is indicated with an arrow (e.g. "Notes on the use of this manual" p. 4). Working steps are numbered. 4 Issue 01 / 2014 innuprep Plant DNA I Kit IPC16

7 Safety precautions 2 Safety precautions Note Read through this chapter carefully prior to guarantee your own safety and a trouble-free operation. Follow all the safety instructions explained in the manual, as well as all messages and information, which are shown. 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. For single use only! This kit is made for single use only! Attention! Don t eat or drink components of the kit! The kit shall only be handled by educated personal in a laboratory environment! If the buffer bottles are damaged or leaking, wear gloves and protective goggles when discarding the bottles in order to avoid any injuries. Analytik Jena AG has not tested the liquid waste generated during using the kit for potential residual infectious components. This case is highly unlikely, but cannot be excluded completely. Therefore, all liquid waste must be considered as potentially infectious and must be handled and discarded according to local safety regulation. Please observe the federal, state and local safety and environmental regulations. Observe the usual precautions for applications using extracted nucleic acids. All materials and reagents used for DNA or RNA isolation should be free of DNases or RNases. Attention! Do not add bleach or acidic components to the waste after sample preparation! Note Emergency medical information in English and German can be obtained 24 hours a day from: Poison Information Center, Freiburg / Germany Phone: +49 (0) For more information, please ask for the material safety data sheet (MSDS): innuprep Plant DNA I Kit IPC16 Issue 01 /

8 Storage conditions 3 Storage conditions The innuprep Plant DNA I Kit - IPC16 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. For further information see table kit components ( "Kit components" p. 7). 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 each innuprep Plant DNA I Kit - IPC16 were tested by isolation of genomic DNA from different plant samples, following spectrophotometric measurement of DNA yield as well as ratio A 260:280 and subsequent amplification of a plant specific target gene. 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 Plant DNA I Kit - IPC16 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. 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! 6 Issue 01 / 2014 innuprep Plant DNA I Kit IPC16

9 Kit components 6 Kit components Important Store lyophilized Proteinase K at 4 C! Divide dissolved Proteinase K into aliquots and storage at 20 C is recommended. Repeated freezing and thawing will reduce the activity dramatically! Storage conditions All other components are stored at room temperature IP_ IP_ IP_ Lysis Solution SLS 15 ml 60 ml 250 ml Lysis Solution OPT 15 ml 60 ml 250 ml Lysis Solution CBV 15 ml 60 ml 250 ml Proteinase K For 2 x 0.3 ml working solution For 2 x 1.5 ml working solution For 7 x 1.5 ml working solution Precipitation Buffer 2 ml 15 ml 2x 30 ml P Pre-Filter (lavender) 16 2x 48 10x 48 Receiver Tube (2.0 ml) 16 2x 48 10x 48 Reagent Strips* 16 (pre-filled, sealed) Reagent Plates* 2 (pre-filled, sealed) 96 (pre-filled, sealed) 12 (pre-filled, sealed) (pre-filled, sealed) Elution Buffer 2 ml 2 ml 2 ml Filter Tips 2x 16 2x 96 10x 96 Elution Tubes (0, x 48 10x 48 ml) Elution Caps x 12 (Stripes) Elution Stripes (0, x 12 ml) Manual Initial steps Dissolve Proteinase K by addition ase K by addition ase K by addition Dissolve Protein- Dissolve Protein- of 0.3 ml of ddh 2 O, mix thoroughly and store as described below! of 1.5 ml of ddh 2 O, mix thoroughly and store as described below! of 1.5 ml of ddh 2 O, mix thoroughly and store as described below! * Depending of order. innuprep Plant DNA I Kit IPC16 Issue 01 /

10 Recommended steps before starting 7 Recommended steps before starting! Important Invert the Reagent Strips for 3 4 times and thump it onto a table to collect the pre-filled solutions at the bottom of the strips. Heat thermal mixer or water bath at 65 C Ensure that the Proteinase K has been prepared according to the instruction ( "Kit components" p. 7) 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 1.5 ml tubes ddh 2 O for dissolving Proteinase K Lysis Tube P (Order no: 845-CS , -100, -250) 8 Issue 01 / 2014 innuprep Plant DNA I Kit IPC16

11 Product specifications 9 Product specifications 1. Starting material: Plant samples fresh, frozen or dried (up to 100 mg dry weight or 180 mg wet weight) 2. Time for isolation: Homogenization: Homogenizer: approx: 30 sec 3 min Liquid nitrogen: approx: 5 10 min Lysis: approx: min InnuPure C16 protocol standard: approx: 47 min InnuPure C16 protocol fast: approx: 38 min 3. Typical yield: Not determined. Depending on type and amount of the starting material. innuprep Plant DNA I Kit IPC16 Issue 01 /

12 Principle of operation 10 Principle of operation Pre-filled reagent structure External lysis Mixing of sample + magnetic particles Transfer of binding buffer to the sample Mixing of sample and binding buffer Collection of the magnetic particles and transfer of the excess Reagent structure The reagent strips or reagent plates are completely pre-filled and contain in addition to the magnetic particles all reagents necessary for the extraction process for the binding, washing and elution of nucleic acids. After starting an extraction protocol the InnuPure C16 takes the tips provided from the corresponding row in the tip block of the sample tray. Lysis The lysis step for the corresponding source material has been described in detail in this manual for the respective extraction kit. Binding With the aid of the binding buffer the nucleic acids are bound to the magnetic particles. In this kit the binding of the nucleic acids takes place in cavity 1 of the reagent structure. In the external lysis the mix of lysed sample and magnetic particles is first homogenized by pipetting on and off. The binding buffer is then transferred to the sample. During the next step the magnetic unit is moved to the floor of the work cavity. The magnetic particles with the bound nucleic acids are held by the magnetic field on the floor of the work cavity. The excess of binding buffer is transferred to the original holding position by pipetting off. 10 Issue 01 / 2014 innuprep Plant DNA I Kit IPC16

13 Principle of operation Washing buffer transfer Washing Collection of the magnetic particles and transfer of the excess Washing of the nucleic acids The magnetic particles remain in the work cavity and the washing buffer is then transferred there. By pipetting different washing buffers on and off the nucleic acids are washed. The number of washing steps and volume and type of washing buffer depend on the type of the source material used. In addition, the buffers differ in the case of DNA or RNA extraction. Between each washing step the magnetic particles are collected with the bound nucleic acids from the cavity floor. The corresponding wash excesses are pipetted off and returned to their original holding position. Tip washing To prevent the carry-over of washing solution residue, the tips used are flushed between specific washing steps. Removal of the washing solution from the 1st work cavity Discharge of the washing solution into the 4th work cavity Change of the work cavity Prior to the final washing step the washing solution is removed completely from the 1st work cavity and transferred by pipetting over into the 4th work cavity. The quality of the already washed nucleic acids is significantly improved by the transfer into a clean work cavity. Any lysis residues at the walls are retained in the "dirty" cavity and only the already washed nucleic acids bound to the magnetic particles are transferred on. Ethanol removal The removal of ethanol residue at the magnetic particles and within the samples takes place via a drying step. In this step a heating is activated in the cavity floor causing the ethanol residue to evaporate. innuprep Plant DNA I Kit IPC16 Issue 01 /

14 Principle of operation Transfer of the elution buffer into the work cavity Heating of the elution buffer Elution and collection of the magnetic particles Change of the work cavity Elution The elution volume can be adjusted in the range from 50 to 500 µl and is defined at the start of the protocol. The correspondingly selected volume is removed from the holding position and transferred to the magnetic particles in the work cavity. The elution process represents the separating of the nucleic acids from the magnetic particles. This is improved by heating the elution buffer and mixing well through pipetting on and off. To be able to transfer the eluates free from magnetic particles into the elution vessels the eluate is transferred once again into a new work cavity. There the remaining magnetic particles are finally collected at the cavity floor. Eluate transfer The nucleic acids are now extracted, are transferred into the elution tube and are available for further downstream applications. 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. 12 Issue 01 / 2014 innuprep Plant DNA I Kit IPC16

15 Homogenization and lysis of plant samples 11 Homogenization and lysis of plant samples After harvesting, if plant tissue will not be used immediately, it can be stored in liquid nitrogen, lyophilized/dried or frozen. Fresh material can be kept at 4 C for 24 hours but should be frozen at - 20 C or for longer storage at - 80 C for later processing. Ground tissue powder can also be stored at - 80 C. Alternatively, tissue can be dried or lyophilized after harvesting to allow storage at room temperature (15 25 C). To ensure DNA quality, samples should be completely dried within 24 hours of collection. We recommended to collect young materials (e.g., leaves, needles) since they contain more cells per weight and therefore result in higher yields. In addition, young leaves and needles contain smaller amounts of polysaccharides and polyphenolics and are therefore easier to handle. Complete and quick disruption of starting material is essential to ensure high DNA yields and to avoid DNA degradation. The lysis procedure is most effective with well-homogenized, powdered samples. Suitable methods include any type of commercial homogenizers (rotor-stator homogenizer) or bead mills (e.g. SpeedMill PLUS, Analytik Jena AG) using ceramic beads. However, we recommend grinding with a mortar and pestle in the presence of liquid nitrogen to obtain optimal yields. When using tissues other than leaves, the disruption method may require optimization to ensure maximum DNA yield and quality. After homogenization and treatment of the sample with lysis solution, the crude lysate can be cleared easily either with Prefilters or by centrifugation. Disruption of starting material using a mortar and pestle Use mortar and pestle to grind the plant material in the presence of liquid nitrogen. Freeze plant material in liquid nitrogen and be careful during homogenization, because do not let the sample thaw at any time. We recommend to precool the using laboratory equipments. Grind frozen plant sample to a fine powder and refill mortar with liquid nitrogen to keep the sample frozen, if necessary. Use precooled tubes for sample storage until lysis step, but make sure no liquid nitrogen is transferred or all nitrogen has evaporated before closing the tube. Disruption of starting material using bead mill homogenizers Use 0,5 g ceramic beads (e.g. 2,4 2,6 mm ceramic beads Lysis Tube P, Analytik Jena AG) for plant material and leaves or 4-5 steel beads in a mixture (e.g. 4,7 mm diameter steel beads Lysis Tube Z, Analytik Jena AG) for seeds, rice and needles. Pipette 50 µl ddh 2 O to the plant material and vortex for about 30 seconds (e.g. SpeedMill PLUS, Analytik Jena AG). Repeat the chilling and vortexing procedure until the entire plant material is ground to a fine solution. innuprep Plant DNA I Kit IPC16 Issue 01 /

16 Homogenization and lysis of plant samples It is also possible to chill the tube in liquid nitrogen. After the homogenization, describe above, chill the tube once more and remove the beads by rolling them out gently or using a magnet. Keep the material frozen throughout the whole homogenization procedure. Do not add nitrogen to the tube since this leads to sticking and loss of plant material attached to the beads. Disruption of starting material using a Rotor-stator homogenizer Rotor-stator homogenizers are only useful to disrupt soft plants in the presence of lysis solution. Keep homogenizer submerged at all times to reduce foaming. Lysis of plant samples Increasing the amount of starting material The standard protocols of innuprep Plant DNA I Kit IPC16 allow processing of mg (dry weight) or mg (wet weight) of plant material. This usually yields 1 25 μg of high quality DNA. However, the amount of DNA that can be expected per mg of sample depends on the size and ploidy of the genome. To optain sufficient DNA yield, it might be advantageous to process a higher than the recommended sample mass. However, to ensure a complete lysis, all lysis solution volumes of protocol step 2 have to be increased proportionally and require multiple loading steps. Selecting the optimal lysis solution system Plants are very heterogeneous and contain varying amounts of polyphenols, acidic components, or polysaccharides which can lead to suboptimal DNA extraction or performance in downstream applications. Therefore, three different lysis solutions are provided for optimal processing, purification performance, high yields and an excellent DNA quality for the most common plant species. The standard protocol uses Lysis Solution SLS, containing CTAB as detergent component. Additionally, the SDS based Lysis Solution OPT is provided which requires subsequent precipitation step to remove all impurities by Precipitation Buffer P. For some plant species a third Lysis Solution CBV leads to higher yield than the both other Lysis Solutions. Lysis Solution CBV requires a subsequent precipitation step, too. If the different Lysis Solutions show similar results in DNA yield and quality please make a choice for the easiest protocol. In order to find optimal lysis conditions when using a certain plant sample for the first time, it is recommended to do side-by-side preparations of one batch of homogeneously ground material with 14 Issue 01 / 2014 innuprep Plant DNA I Kit IPC16

17 Homogenization and lysis of plant samples the three different lysis solutions and make a decision for the best one (regarding yield, quality or other relevant parameters). The following example of application illustrates the effects of different lysis solutions on yield and quality of the extracted gdna. Application example: Isolation of gdna from parsley (Petroselinum grispum) using the three different lysis solutions SLS, OPT and CBV. The spectrophotometric measurement shows different results depending on the lysis solution used. Fig. 1: Concentration of DNA with Lysis Solution SLS: 16.8 ng/µl and purity (A 260 /A 230 ): Fig. 2: Concentration of DNA with Lysis Solution OPT: 7.0 ng/µl and purity (A 260 /A 230 ): Fig. 3: Concentration of DNA with Lysis Solution CBV: 59.0 ng/µl and purity (A 260 /A 230 ): Important note For a large variety of plant species, either lysis solution generates good results. innuprep Plant DNA I Kit IPC16 Issue 01 /

18 Protocols: DNA isolation from plant material 12 Protocols: DNA isolation from plant material Note The lysis of the starting material is a preliminary manual processing step. Please note that up to 100 mg of plant material can be processed. To maximize the final yield of DNA a complete homogenization of plant sample is important! Important note The innuprep Plant DNA I Kit IPC16 include three different lysis solutions for optimal results with most common plant species. Please refer to section Homogenization and lysis of plant samples (p. 13) for choosing the optimal lysis solution system for your individual plant sample. 1. Homogenization of about mg of starting material by a pestle under liquid N 2. Commercially available equipment for homogenization (e.g. SpeedMill PLUS) also can be used. Alternatively grinding of plant material with sand is also possible ( Homogenization and lysis of plant samples, p. 13). Note: Use mg of starting material if extraction from material which is very wet or contains more water. Proceed with cell lysis using Lysis Solution SLS (Protocol 1), Lysis Solution OPT (Protocol 2) or Lysis Solution CBV (Protocol 3) Protocol 1: gdna from plant using Lysis Solution SLS 2. Transfer the plant powder or other homogenized starting material in a 1.5 ml or 2.0 ml reaction tube. Add 500 µl Lysis Solution SLS and 20 µl Proteinase K, mix vigorously by pulsed vortexing for 5 sec. Incubate at 65 C for approx 30 minutes (longer incubation is also possible, up to 60 min). 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 3 4 times during lysis step. No shaking will reduce the lysis efficiency. 3. Transfer the sample onto a Prefilter (lavender) located in a 2.0 ml Receiver Tube and centrifuge the tube at x g ( rpm) for 1 minute. Discard the Prefilter. Don t discard the 2.0 ml Receiver Tube with the filtrate! 16 Issue 01 / 2014 innuprep Plant DNA I Kit IPC16

19 Protocols: DNA isolation from plant material Note: Don t discard the pellet at the ground of the 2.0 ml Receiver Tube. Note: To remove RNA from the sample (if necessary) add 4 µl of RNase A solution (100 mg/ml) to the filtrate, vortex shortly and incubate for 5 min at room temperature. 4. Transfer 400 µl of the filtrate into the first cavity of Reagent Strips or Reagent Plates (Optional: Avoid a carry-over of solid material/pellet). ( "Sample", p. 22) Note The lysed sample will be processed using the InnuPure C16. Please follow the instruction of the manual from point 13! 12.2 Protocol 2: gdna from plant using Lysis Solution OPT 2. Transfer the plant powder or other homogenized starting material in a 1.5 ml or 2.0 ml reaction tube. Add 500 µl Lysis Solution OPT, mix vigorously by pulsed vortexing for 5 sec. Incubate at 65 C for approx 30 minutes (longer incubation is also possible, up to 60 min). 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 3 4 times during lysis step. No shaking will reduce the lysis efficiency. 3. Add 100 µl Precipitation Buffer P and vortex the sample for 5 sec. Incubate at room temperature for 5 min and centrifuge at maximum speed for 5 min. 4. Transfer the clear supernatant onto a Prefilter (lavender) located in a 2.0 ml Receiver Tube and centrifuge the tube at x g ( rpm) for 1 minute. Discard the Prefilter. Don t discard the 2.0 ml Receiver Tube with the filtrate! Note: Don t discard the pellet at the ground of the 2.0 ml Receiver Tube. Note: To remove RNA from the sample (if necessary) add 4 µl of RNase A solution (100 mg/ml) to the filtrate, vortex shortly and incubate for 5 min at room temperature. 5. Transfer 400 µl of the filtrate into the first cavity of Reagent Strips or Reagent Plates (Optional: Avoid a carry-over of solid material/pellet). ( "Sample", p. 22) Note The lysed sample will be processed using the InnuPure C16. Please follow the instruction of the manual from point 13! innuprep Plant DNA I Kit IPC16 Issue 01 /

20 Protocols: DNA isolation from plant material 12.3 Protocol 3: gdna from plant using Lysis Solution CBV 2. Transfer the plant powder or other homogenized starting material in a 1.5 ml or 2.0 ml reaction tube. Add 500 µl Lysis Solution CBV and 20 µl Proteinase K, mix vigorously by pulsed vortexing for 5 sec. Incubate at 65 C for approx 30 minutes (longer incubation is also possible, up to 60 min). 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 3 4 times during lysis step. No shaking will reduce the lysis efficiency. 3. Add 100 µl Precipitation Buffer P and vortex the sample for 5 sec. Incubate at room temperature for 5 min and centrifuge at maximum speed for 5 min. 4. Transfer the clear supernatant onto a Prefilter (lavender) located in a 2.0 ml Receiver Tube and centrifuge the tube at x g ( rpm) for 1 minute. Discard the Prefilter. Don t discard the 2.0 ml Receiver Tube with the filtrate! Note: Don t discard the pellet at the ground of the 2.0 ml Receiver Tube. Note: To remove RNA from the sample (if necessary) add 4 µl of RNase A solution (100 mg/ml) to the filtrate, vortex shortly and incubate for 5 min at room temperature. 5. Transfer 400 µl of the filtrate into the first cavity of Reagent Strips or Reagent Plates (Optional: Avoid a carry-over of solid material/pellet). ( "Sample", p. 22) Note The lysed sample will be processed using the InnuPure C16. Please follow the instruction of the manual from point 13! 18 Issue 01 / 2014 innuprep Plant DNA I Kit IPC16

21 Preliminary steps of the InnuPure C16 13 Preliminary steps of the InnuPure C Sample tray No. 1: No. 2: No. 3: No. 4: No. 5: No. 6: No. 7: 13.2 Filter tips Elution vessels for purified samples Tip block Pressure pad Sample block for reagent plates or adapter for reagent strips Serrated guide rail Adapter for reagent strips Preparing sample tray Note The needed number of reagent strips or reagent plates is depending on the number of samples, which have to be processed. Don't use more strips as number of samples! Invert the reagent strips or reagent plates for 3 4 times and thump it onto a table to collect the pre-filled solutions at the bottom of the single strips or reagent plates. innuprep Plant DNA I Kit IPC16 Issue 01 /

22 Preliminary steps of the InnuPure C16 Move the InnuPure C16 sample tray into the priming station and fold the holding-down clamp at the sample tray upwards! Place the reagent plate into the holder of the sample tray. The red line on the cover film must point to the red dot at the holder. Place the adapter for the reagent strips into the holder. Here, too, the red marking on the adapter must point to the red point at the holder. Caution: Load the sample tray evenly in both adapters (see below)! Place the strips into the adapter. The long tab marked with the label "AJ" must point to the engraved numbers on the sample tray. 20 Issue 01 / 2014 innuprep Plant DNA I Kit IPC16

23 Preliminary steps of the InnuPure C16 Fold down the holding-down clamp to prevent the plates and strips to be pulled out of the holder during the extraction process. Place the filter tips into the two rows with the smaller drill holes. Place the elution vessels into the wider drill hole at the edge of the tip block. Empty sample positions do not need to be filled. Especially with the strips make sure that for every strip the tips and the elution vessel are in the corresponding positions in the tip block (arrows in figure on the left)! Important Note It is possible to select between two different elution vessels! For smaller elution volume (up to 200 µl) use Elution Stripes (0,2 ml) and for higher elution volume (up to 500 µl) use Elution Tubes (0,65 ml) with corresponding Elution Caps (Stripes). innuprep Plant DNA I Kit IPC16 Issue 01 /

24 Preliminary steps of the InnuPure C Sample Note The following step will be done after the sample lysis! 1. Open all cavities of positions 1 and 3 on the strips and plates by piercing the film with an unused filter tip and widening it or using the optionally available perforation tool (see item below). For the strips, position 1 is at the tab labeled "AJ". On the plates the position numbers are attached on the side.! Important - Caution Open the cavities fully (at least 7 mm in diameter)! The used filter tips are ejected into these cavities during the extraction protocol. If the cavities are closed or insufficiently opened, the tips may be thrown out of the cavities after ejection due to travel. This may result in errors during the extraction process and in adverse cases even a cancellation of the protocol. When using the perforation tool, place it onto the edges of the rear holding-down clamp with the four long bolts sitting against the edges. Then press the tool down until the stop to make the tips pierce through the sealing film and creating large openings. The use of the tool is identical for reagent plates and strips. 2. Transfer the lysed sample into the first cavity of Reagent Strips or Reagent Plates, as shown below. Transfer the sample into the first cavity of reagent strips or reagent plates. If reagent strips are use, the cavity behind the label "AJ" is the first cavity use the first cavity for transfer the sample, if reagent plates are use, the cavity 1A to 1H are the first cavities. 22 Issue 01 / 2014 innuprep Plant DNA I Kit IPC16

25 Automatic processing of the InnuPure C16 14 Automatic processing of the InnuPure C16 1. Switch on the InnuPure C16 and wait for the device initialization to complete, which is signaled by a beeping sound. 2. Move the loaded sample tray with the reagent strips forward into the adapter on the front of the InnuPure C16. The serrated rails at the side of the sample tray must protrude into the grooves of the adapter. After pressing lightly against the tip block the sample tray is automatically pulled into the device.! Important Caution Risk of crushing Immediately let go of the sample tray once it is being pulled in. Otherwise there is a risk of your hand being crushed. 2. Start the extraction protocol: Important Note It is possible to select between two different extraction protocols! A standard protocol DNA_external_Lysis_C16_02 (47 min) and a fast protocol DNA_external_Lysis_FAST_C16_02 (38 min). Please test which protocol is the best for your specific application. In the start window press the button [SELECT PROTOCOL] Select the desired extraction protocol DNA_external_Lysis_C16_02 or DNA_external_Lysis_FAST_C16_02 and press [START] Enter the recommended eluate volume of 200 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 C16 Handbook Starting an extraction protocol on page 30! innuprep Plant DNA I Kit IPC16 Issue 01 /

26 Automatic processing of the InnuPure C16 4. After completion of the protocol press again lightly against the tip block. The sample tray is then automatically moved out of the sample block. Note: The chosen protocol is performed by device and after the protocol is finished, the tray with the purified samples will be moved out and the message 'Program finished' is shown in the screen of the device! 5. Remove the sample tray from the adapter of the InnuPure C16 and move it back into the priming station. 6. After finishing the extraction protocol, the Elution Stripes (0,2 ml) or Elution Tubes (0,65 ml) on position 2 contain the extracted DNA; close the lids and store the DNA under proper conditions. Close the elution vessels. Note: Be careful when closing and store the elution vessels.! Note Store the DNA under adequate conditions. We recommend to store the extracted DNA at 20 C! Important For some downstream applications we recommend a dilution of the extracted DNA with water. 24 Issue 01 / 2014 innuprep Plant DNA I Kit IPC16

27 Related Products 15 Related Products Name Amount Order No. Nucleid acid purification innuprep Proteinase K 6 mg 845-CH mg 845-CH Products for PCR & Gel Electrophoresis innutaq DNA Polymerase (5 U/µl) 500 U 845-EZ innutaq RED DNA Polymerase (1 U/µl) 500 U 845-EZ innutaq Hot-A DNA Polymerase (5 U/µl) 500 U 845-EZ innutaq UltraPure DNA Polymerase (5 U/µl) 500 U 845-EZ x innucleotide Mix (12,5 mm) 2x 0.5 ml 845-AS innucleotide Set (100 mm) 4x 0.25 ml 845-AS mm MgCl 2 - Solution 3x 1.5 ml 845-AS mm MgCl 2 - Solution 3x 1.5 ml 845-AS PCR-grade H 2 O 2.0 ml 845-AS x 2.0 ml 845-AS innumix rapidpcr MasterMix 100 rxn 845-AS rxn 845-AS innumix Standard PCR MasterMix 100 rxn 845-AS rxn 845-AS innumix Green PCR MasterMix 100 rxn 845-AS rxn 845-AS innustar 100 bp DNA Ladder Express 500 µl 845-ST x 500 µl 845-ST innustar 1 kb DNA Ladder Express 500 µl 845-ST x 500 µl 845-ST x Loading Dye Bromophenol Blue 3x 1.0 ml 845-ST x 1.0 ml 845-ST x Loading Dye Orange G 3x 1.0 ml 845-ST x 1.0 ml 845-ST Products for qpcr innumix qpcr MasterMix Probe 100 rxn 845-AS rxn 845-AS innumix qpcr MasterMix SyGreen 100 rxn 845-AS rxn 845-AS innuprep Plant DNA I Kit IPC16 Issue 01 /

28 engl. /1 Analytik Jena AG, Jena

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