Thank you for purchasing Kilobaser, a personal bench-top DNA synthesizer.
With Kilobaser you can quickly and reliably produce DNA primers and accelerate your DNA workflows. The use and handling is simple and intuitive, Kilobaser is intended to automatically perform the entire production of the DNA, from synthesis itself to the post-synthetic operations.
No special training in reagent handling is needed, since all reagents are contained in the patented close-loop cartridge system. The synthesized DNA is dried in the collection vial and thus still contains salts, protective groups and unreacted phosphoramidites and can be directly used for most of the applications.
Kilobaser is not classified as medical device. The produced DNA is intended for research and development purposes only, not for for medical use .
In order to use and maintain the device properly, please read the manual thoroughly and keep it for future reference. Not following the instructions can lead to damage of the device, reagent contamination and in worst cases to personal injuries.
The present instruction manual is valid as per current state. You will be informed about the future updates, that will be available to download from the website: https://manual.kilobaser.com
Name and Address of the Manufacturer
|Company Name||Kilobaser GmbH|
|Street Address||Reininghausstrasse 13a|
|Country||Austria / Europe|
|Time of synthesis per base||2.5 min|
|Post-synthesis treatment time||30 min|
|Time of the activation of the cartridge||20 min|
|Maximal length of DNA||80 bases|
|Supply gas||Argon with purity of at least 99.999 %|
|Supply gas pressure||6 to 10 bar (87 to 145 psi)|
|Mains supply||100-240 VAC|
|Input voltage||24 VDC|
|Input current||8 A|
|Connection||cable with a supplied 24 V power supply|
|Remote inputs||USB, HDMI, RJ45|
|Dimensions||HxWxD 32.5x28x31.5 cm|
|Environmental||Indoor use only|
|Ambient temberature||15 to 40 °C (non condensing)|
|Humidity||0 to 60 % relative humidity|
Inside – Lid Open
Consumables, which are sold by Kilobaser separately, include microfluidic chips, reagent cartridges and collection vials. Microfluidic chips and reagent cartridges suitable for Kilobaser are exclusively produced and sold by Kilobaser and its official distributors. Do not use any unofficial consumables you may encounter and please help avoiding injury and equipment destruction by notifying us if you encounter any unofficial Kilobaser consumables.
Our single-use microfluidic synthesis chips are multi-layer transparent plastic chips containing a single on-chip synthesis column and an array of microfluidic membrane valves. DNA synthesis works by attaching one nucleobase after another in a cyclic process, starting from linker molecules on the column surface. After the synthesis, the linker molecules are cleaved and DNA is eluted into the DNA collection vial. Since the linker molecules on the column surface are destroyed during cleavage, chips cannot be reused. Please make sure you mark or discard used chips immediately to prevent accidental reuse! If you cancel an operation (see Canceling operations) immediately after start, chips can be reused up to a certain point. Kilobaser will instruct you how the handle the inserted chip after cancel.
The Kilobaser reagent cartridge contains all reagents necessary for all operations of Kilobaser. No additional solvent handling is needed. Liquid waste is collected in the waste sections of the cartridge.
Hazard warning! Although Kilobaser’s liquid handling system is designed to eliminate user exposure to hazardous reagents, the cartridge still contains dangerous chemicals which can be released by accidents or severe mishandling. In case of emergency, please immediately ventilate and clear the area and consult the Material Safety Data Sheet (MSDS) and/or customer support for further instructions!
It is not possible to change cartridges in the middle of a (long) synthesis operation. A single cartridge has a capacity of roughly 200 bases, assuming a mean oligo length of 20 bases. Since every synthesis operation requires a basal reagent amount, you can produce a slightly higher number of bases if your oligo length is longer. Once a cartridge is inserted and activated, it needs to be consumed within 14 days (336h).
Your distributor is required to manage the recycling process with respect to your local circumstances. Usually, used cartridges will be sent back to or collected by your distributor without additional handling steps required. If further handling (e.g. disassembly and separate disposal of cartridge contents) is necessary, please adhere to the instructions given by your distributor.
The collection vials supplied by Kilobaser are high quality 200μL PCR vials suitable for direct use in most thermocyclers, including qPCR appliances. You are free to use other vials at your own risk, as long as they fit into the heated DNA collection port and allow good heat transmission. If you experience any problems like remaining liquid (droplets) or deformed shape, please use vials supplied by Kilobaser.
Warnings and safety precautions
Keep out of the reach of children.
Observe the ambient conditions mentioned in Technical data.
Please do not put any objects atop of the Kilobaser.
Do not move Kilobaser when in operation.
Do not expose Kilobaser to corroding influences.
Do not expose Kilobaser to or clean it with chemicals and solvents, except where stated otherwise (see Cleaning and maintenance).
Please note that there can be reagent remnants left under the chip when the synthesis is finished and the lid is opened. Wear protective gloves when touching the used chip!
Please note that crystalline rests can be formed on the rubber nubs, onto which the chip is placed (see Cleaning and maintenance).
Please note that when taking the cartridge out of the Kilobaser (see Inserting a cartridge), the septa on the reagent bottles are pierced and so not completely sealed anymore. In some cases, small amounts of reagents can come out. Wear protective gloves when handling with a used cartridge! Keep a used cartridge upright until disposing of it (see Cartridge recycling disposal).
After synthesis, the heating block can still be very hot! Please be careful when removing the vial with your DNA! The best way to remove the vial is with the help of pincers.
Please note that there can be unpleasant smell coming out from the exhaust gas outlet during the last steps of the synthesis. Place Kilobaser in a well-ventilated area. If smell is too Irritating, use an exhaust gas filter cartridge supplied by Kilobaser (not included) (see Exhaust gas).
Please be aware that Kilobaser is a heavy object (21 kg); extra care should be taken by lifting and carrying. Be careful not to drop the Kilobaser.
Even though Kilobaser is considered as a safe device and has integrated safety mechanisms to prevent damage and injuries, please take extra care during steps where the motors are moving!
Although Kilobaser’s liquid handling system is designed to eliminate user exposure to hazardous reagents, the cartridge still contains dangerous chemicals. These can in very small amounts appear under the used chip after a synthesis. Wear protective gloves! By accidents or severe mishandling, reagents can be released in bigger amounts. In case of emergency, please immediately ventilate and clear the area and consult the Material Safety Data Sheet (MSDS) and/or customer support for further instructions!
If damage is detected, stop using Kilobaser immediately and contact the Kilobaser support.
Do not attempt to open or repair Kilobaser yourself, unless if instructed otherwise. Repairs can only be performed by qualified personnel or manufacturer.
ONLY ORIGINAL PARTS AND CONSUMABLES FROM THE MANUFACTURER MUST BE USED. USING THIRD PARTY OR NON ORIGINAL CONSUMABLES WILL RESULT IN DAMAGE TO THE MACHINE AND VOID THE WARRANTY!
Unpacking and inspection
Prior to opening the shipping container, inspect it for damage or evidence of mishandling. If it has been damaged or mishandled, notify the carrier before opening the container. Carefully unpack the Kilobaser and accessories and inspect them for completeness or possible transport damages. Any damage should be reported to the carrier and to the supplier immediately. Save the shipping container. Check the contents against the packing list (see Scope of delivery).
Scope of delivery
- Power supply with cables, packaged separately
- Display on the display stand
- USB-C cable
- HDMI cable
- Instruction manual
Place the Kilobaser on a stable, flat and dry surface, keeping in mind its weight. There should be at least 15 cm clearance in the back for proper ventilation and the necessary electrical and gas connections, as well as at least 25 cm clearance above to allow for unconstrained opening of the lid. The acceptable environment for Kilobaser is normal indoor laboratory conditions. The area must be clean and well-ventilated; preferable location is a fume hood, where a good suction can remove gases, that are released during the drying step (see DNA Synthesis). Temperature should be stable and humidity should not be too high, as the DNA synthesis is moisture-sensitive. The specific temperature and humidity conditions are 15 to 40 °C and 0% to 60% relative humidity.
Connect the Kilobaser to a 100-230 VAC, 50-60 Hz power socket using the supplied external power supply unit. Input voltage is 24 V/8 A.
Take care to connect the power supply to Kilobaser first, before connecting the power supply to the wall outlet.
Kilobaser is equipped with a separate, movable touch screen display connected via HDMI/USB cables. It allows easy access to local operations without login credentials, as well as configuration of remote connection and integration into your network via Ethernet or WiFi. You can also start synthesis operations for sequences either prepared by remote access from your PC or entered directly on the touch screen. If you do not want to use the touch screen or have several devices located next to each other, Kilobaser can also be completely remotely controlled via its web interface.
For Kilobaser to function properly, an argon gas source with pressure between 6 and 10 bar must be connected to the push-in connector on the Kilobaser using a 6 mm O.D. flexible pneumatic tubing. To connect the Kilobaser to a gas supply, the following is required (not supplied by Kilobaser):
- Gas source
- Pressure regulator
- Pneumatic fittings
- Pneumatic tubing
While in principle any inert gas is suitable, it is highly recommended to use argon with purity of at least 99.999 % (≤2ppm O~2~ and ≤3ppm H~2~O). Connecting less pure argon or nitrogen may result in higher moisture in the system and consequently in decreased purity of the synthesized DNA. Many laboratories receive an inert gas from a central gas supply through pipelines, while some have a safety cabinet where pressurized gas cylinders are kept. If there is no central gas supply and no safety cabinet in the laboratory, keep in mind that compressed gas cylinders must be stored upright and according to hazard classification in a well-ventilated, above-grade, weather-proof storage area that is a safe distance from combustible materials, ignition sources, or intense heat. Gas cylinders can be obtained in several different sizes. For an idea: a 50 L, 10 L or 1 L compressed argon cylinder (all 200 bar) will suffice for approximately 1000, 200 or 20 syntheses, respectively. While gas cylinders can be bought from numerous suppliers, we recommend a dealer that offers services such as refill, exchange and pickup of empty gas cylinders.
In case of a central gas supply, outlet points are usually fitted at the end of the pipeline to set pressure. Safety cabinets are normally equipped with pressure control panels. In a situation where a pressure regulator is not available in the laboratory, it is important to get a proper one since there are many different kinds on the market. For example, a pressure regulator for welding is not suitable. For argon, only a pressure regulator for argon is fitting. It should be equipped with a pressure gauge with a display range to at least 10 bar (145 psi). Setting an appropriate pressure is very important. It has to be set between 6 and 10 bar (87 and 145 psi). A lower pressure will cause Kilobaser to stop preventively to protect the cartridge. Since standard pneumatic tubing and fittings are usually pressure resistant up to 10 bar, a higher pressure may result in gas leakage.
Whether it is at outlet of a pipeline, of a pressure regulator or of a fume hood gas fitting, the type of the outlet connection may be very different. Kilobaser has a push-in connector to fit a 6 mm O.D. pneumatic tubing, so it is important that the 6 mm tubing can be connected to the outlet connection of the source. If the tubing cannot be fitted directly, a suitable pneumatic fitting or connector is needed. One side of the pneumatic connection of the fitting has to be a push-in type for a 6 mm O.D. pneumatic tubing, while the other side has to fit onto the outlet connection of the source. The outlet of a pressure regulator is normally equipped with a nozzle with a thread, which can be screwed off and exchanged for a suitable pneumatic fitting (one side the appropriate thread that fits the pressure regulator outlet, and the other side a push-in type for a 6 mm O.D. pneumatic tubing).
The gas source and Kilobaser are connected with a 6 mm O.D. flexible pneumatic tubing.
During the final steps of the post-synthetic treatment a solvent is evaporated from the collection vial (see Synthesis). During this step gases with an unpleasant smell can be released from the exhaust gas outlet. Place the Kilobaser to a well-ventilated area, preferably a fume hood, to suck away the released gases. If the smell is too irritating, an exhaust gas filter cartridge supplied by Kilobaser (not included) can be connected to the push-in outlet. Please note that it is recommended to replace the filter cartridges once a year or as soon as an irritating smell occurs again.
Before your first synthesis
Kilobaser is shipped without any consumables, that is without a microfluidic chip, a collection vial and a reagent cartridge (see Consumables). Make sure that before the first synthesis an unused chip, a clean vial and a new reaction cartridge are available. Do not start a synthesis if one of the consumables is not in stock. If a synthesis is nonetheless accidentally started before realizing that one of the consumables is not available, the run can be canceled and returned to the initial state (see Canceling operations).
Switching on Kilobaser
To switch on, press the on/off button in the front side of the Kilobaser. In a couple of seconds the screen shows the user interface.
Synthesis operations are prepared by sending a DNA sequence to Kilobaser. This can be accomplished by several means:
a) direct input using the touch screen or remote interface, b) copy&paste from your PC using the remote web interface or.
Please apply usual guidelines for DNA sequence/PCR primer design also for Kilobaser.
Metadata like name of the project or description can be assigned to each sequence. It is also possible to define additional metadata fields to save data like project identifiers, analysis results or inventory (e.g. LIMS) identifiers. This is done by clicking on the “Edit current project” button and choosing “Edit metadata fields”. By clicking on the icon “Edit fields” you can enter the text.
After sending a DNA sequence to Kilobaser, it is added to a queue. There you can view all the information concerning the current sequence, for example the duration of the synthesis (always time of the entire synthesis, from initialization to dry product) and expected concentration at a resuspension volume of 10 µL.
It is possible to add optional number of DNA sequences, wherein they will be queued in order of creation. A sequence can also be deleted from the queue by choosing “Delete from queue” in the “Edit current project” menu. If a cartridge change is required before the next sequence (see Inserting a cartridge), this is shown in the queue with a “Cartridge change” symbol.
A current state of a cartridge can be checked at any time by clicking on the “Cartridge status” symbol. The activation date is shown, as well as the expiration date, which is 14 days after the activation of the cartridge. Kilobaser predicts the expected reagent consumption of your planned synthesis operations and instructs at which point in the queue the cartridge needs to be replaced. This allows you to plan sequences to make the best use of the remaining bases of a cartridge. The number of remaining oligos is calculated in the case where synthesized oligos are 20-mers. Please note that the number of remaining bases is only an approximation for you too have a rough overview. For the exact number of oligos that can be synthesized, the sequences should be added to the queue. Please don't start the run if you have no new cartridges in stock!
The sequence that is listed in the queue at the very top, is the next to be synthesized. Synthesis is started by clicking on the “Start synthesis” button.
DNA synthesis, which includes synthesis on a chip, cleavage from the chip, deprotection at an elevated temperature and drying, occurs completely automatized. It is, however, necessary that the user inserts/changes the consumables (see below). During the user interaction, the motors have to be moved to open/close the lid (see Lid open) or to eject/insert the cartridge (see Inserting a cartridge). Kilobaser will never move the motors automatically without your confirmation! On the display it will be clearly instructed when a certain interaction is required.
As mentioned above, Kilobaser is shipped without consumables. After starting the very first synthesis and before cartridge insertion, system is automatically purged for 2 min with argon gas to remove any moisture from the system. After that, the synthesis proceeds normally with cartridge insertion (see Inserting a cartridge).
After pressing the “Start synthesis” button, Kilobaser will inform you that the lid will be opened. By pressing on the “Confirm” button, the motors start moving and the lid opens. Please make sure nothing is standing on the lid. If the free movement of the lid is obstructed in any way or if you feel that the ongoing opening will cause any kind of damage or injury, the movement can be stopped by pressing the “Stop” button. The motors will stop immediately and will not move again until your further action (see Canceling operations). The lid should not be left open any longer than necessary, since reagents in the cartridge are very sensitive to air humidity!
Inserting a chip
After the lid is opened, you will be asked to insert a microfluidic chip (see Microfluidic chips). The chip is inserted so that it is placed on the rubber nubs and fits into the chip slot, with a truncated vertex on the lower right side. Please be careful to insert a new and unused chip. Used chips should be clearly marked and cannot be reused! Make sure that the chip fits exactly into the slot and does not lie on the edges, otherwise the closing lid can break the chip. If you hear that the chip has cracked, stop the run immediately by pressing the “stop” button (see Canceling operations). In the choice field choose “End process: Finish up/Insert placeholder” and start the synthesis from the beginning with a new unused chip.
Because of the material properties of the rubber sealing gasket on the inside of the lid, a microfluidic chip or a placeholder (see End process: Finish up/Insert placeholder) can stick to its surface and stay attached to it. **Make sure to remove a chip or a placeholder from the gasket! **Not doing so can lead to damage of the chip or Kilobaser and can cause a situation where after closing the lid the system is not gas-tight and can in worst cases lead to spillage of reagents into the system.
Inserting a vial
After you have confirmed that the chip is correctly inserted, you will be instructed to insert a PCR vial (see Collection vial). Insert the PCR vial into the port in the heater block. If you cannot fit your PCR vial into the port, please use the vials supplied by Kilobaser.
Finally, after inserting a chip and a vial, an instruction appears that the lid will be closed. By pressing on the “Confirm” button, the lid starts closing. Make sure there are no objects or body parts obstructing the free movement of the lid. The lid has a safety mechanism, where in case of obstruction it stops and so cannot squeeze body parts or objects. If you feel that the ongoing closing will cause any kind of damage or injury, the movement can be stopped by pressing the “Stop” button. The motors will stop immediately and will not move again until your further action (see Canceling operations).
Inserting a cartridge
When there are no more reagents in the cartridge or the cartridge has expired, Kilobaser will automatically plan a replacement of the cartridge. After confirming that the old cartridge will be moved down, you will be asked to insert a new cartridge. By the very first synthesis, you will be instructed to insert a new cartridge after the system has been purged (see First synthesis).
Make sure that when inserting the cartridge, you push it to the very end of the cartridge slot until you feel that it has been gently locked in place. The cartridge is automatically moved up after you confirm it. Make sure nothing is obstructing the free movement of the cartridge. If you feel like the movement is obstructed, you can stop the motor by pressing the “stop” button. The motor will stop immediately and will not move again until your further action (see Canceling operations).
When a new reagent cartridge is inserted, it is automatically activated. This takes 20 extra minutes, since crystalline reagents need to be diluted.,since solid reagents have to be dissolved After that, synthesis proceeds normally.
When the last step of the synthesis, drying, is finished, a choice field appears where Kilobaser offers you two options:
a) Continue with the next sequence:
This option is only available if there is another sequence in the queue. In this case, synthesis of the next sequence in the queue is started. Make sure you take the vial out of the heating block (see Taking the DNA collection vial out of the heating block) and exchange the chip as well as insert a new vial! Follow the instructions given by Kilobaser. The process proceeds as described from the point “188.8.131.52. Lid open”.
b) End process: Finish up/Insert placeholder:
If you do not want to continue synthesizing, you should select “End process” from the choice filed. By pressing the button the lid is opened and you are instructed to insert a placeholder. Placeholder is a plate with a form of a chip that is responsible for keeping the reagents from spilling out of the cartridge. Please make sure to insert the placeholder the same way as a chip (see Inserting a chip). If a placeholder is inserted, Kilobaser can be switched off safely and the argon bottle can be turned OFF. The next synthesis can of course be started at any time. Don't forget to turn Argon ON again in this case!
Before you select any of the two choices, the lid remains closed. It is safe to keep Kilobaser in this state for a few hours, e.g. overnight (important: do not turn off argon until a placeholder has been inserted! The cartridge and in some cases the whole device can be contaminated on pressure drop).
Taking the DNA collection vial out of the heating block
Whether you choose to continue with the next sequence or finish up, make sure you take out the DNA collection vial with the dried synthesized DNA. You will be reminded to do so by Kilobaser.
Caution: Please be careful when removing the vial from the heating block! The heating block may still be very hot! The best way to remove the vial is with some pincers.
After the synthesis
The DNA vial contains beside the synthesized sequence also salts from the protecting groups, unreacted phosphoramidites and some truncated sequences. For most of the applications, like PCR, no further purification is needed. Re-suspend the dry DNA according to the instructions displayed by the software. You need DNAse/RNAse free water or, preferably, a TRIS-EDTA buffer for re-suspension of dried DNA. Non of the solvents is supplied by Kilobaser. Usually, adding 10μL DNAse/RNAse free water leads to a solution with 25-30 pmol/μL. The exact concentration strongly depends on sequence, length and synthesis quality (which further depends on the environmental conditions like humidity). If you require the DNA desalted and with higher purity, you can use purification columns. If you have more questions about purification, contact Kilobaser for support.
When Kilobaser is not synthesizing, you can open it by choosing “Open device” from the drop-down menu. Carefully follow the instructions given by Kilobaser! Basically, this protocol performs operations “Lid open” and “Lid close”. Make sure that before confirming closing the lid you correctly insert the placeholder (see End process: Finish up/Insert placeholder).
For transportation the cartridge needs to be removed from the device and discarded. This can be accomplished by selecting the “Remove disposables” operation from the drop-down menu. Make sure to carefully follow the instructions given by Kilobaser.
Even though this operation can also be performed if the device is not used for an extended time period, it is recommended to leave the cartridge inserted nevertheless. It can happen that the crystalline reagents crystallize in the needles and block them; the syntheses will consequently not be successful. If you still want to remove disposables, you can do it at your own risk. If you suspect that blockage of the needles has occurred, please immediately contact the Kilobaser for support.
When Kilobaser is working, any step can be canceled at any time. This is done by pressing the red “Stop” button at the top of the screen. Note that during most of the steps of the synthesis pressing the “Stop” button does not yet cancel the current process, it continues to run normally. The run also proceeds normally if you select “No” button from the pop-up window. To cancel the synthesis, select “Yes” button from the pop-up window. In this case, Kilobaser will automatically execute a canceling protocol (please not that this can take up to 90 seconds, depending on in which state the synthetic procedure currently is) and jump straight into the choice field (see Choice field), where you can select to synthesize the next sequence in the queue or finish and insert a placeholder.
There is a slightly different course of canceling for steps where motors are moving. By pressing the “Stop” button, the motors stop immediately. If selecting “No” from the pop-up window, the process will continue, however the lid will always be opened first before continuing. By selecting “Yes” from the pop-up window the process will be canceled: please carefully follow the instructions, given by Kilobaser. After the run is canceled, the Kilobaser will offer the choice field (see Choice field). Make sure that when canceling the movement of the lid, you do not leave it in the paused state for any longer than necessary, since reagents in the cartridge are very sensitive to air humidity!
Note: Cancel mechanisms are subject to ongoing optimization and may not fully work as expected in some edge cases. If you encounter any situation where canceling could be handled smoother we are very glad for your feedback!
Cleaning and maintenance
Special cleaning of the device is not necessary. If you notice white crystals forming on the rubber nubs, onto which the microfluidic chip is placed, you can wipe them with a soft tissue soaked in dry acetonitrile. Do not leave the lid opened for longer than necessary!
Please note that the rubber sealing gasket is not solvent resistant. Do not clean the rubber sealing gasket, as this can impair the DNA synthesis! If you suspect that the rubber sealing gasket is defected, you can attempt to exchange it yourself. Carefully pull the gasket out of the wholes in the aluminum block, taking care that you do not tear off the nibs. After removing the old gasket, a new one can be inserted by firmly pushing the nibs into the wholes. In case that the nibs tear from the gasket surface, this needs to be repaired by a qualified person – please contact Kilobaser support.
If you use an exhaust gas filter cartridge against chemical smell, note that it is recommended to replace them once a year or as soon as an irritating smell occurs again.
Kilobaser is an automated device that does not require any special maintenance. If you notice that operations are not functioning properly or as they should, please contact Kilobaser for support.
|I touched some reagent remnants without nitrile gloves.||Please wash your hands thoroughly. Although the chemicals are considered harmful, the solvents (mostly acetonitrile) evaporate very quickly, leaving either no remnant or harmless salts. In case of bigger spills (e.g. crushed cartridge(s)), immediately ventilate and clear the room and consult the MSDS or customer support for further instruction.|
|I hear a hissing noise after the lid is locked.||If Kilobaser does not recognize the leak automatically, please cancel the operation (see Canceling operations). Check for any defects on the rubber sealing gasket and replace it if necessary. If you can't find the culprit or the noise does not disappear, please contact customer support.|
|DNA does not work in my process.||We are very sorry for your inconvenience! Please consider if there may be any “difficult” sections in your sequence, e.g. leading to secondary structures in your DNA molecule. Consult DNA design guidelines to avoid certain pitfalls like palindromes, hairpins or GC-rich sections. If the problem remains, please consider ordering the oligo from a synthesis service provider. For some processes it can be beneficial to try downstream purification, although yield may drop significantly using typical spin-column kits.|
|The touchscreen display looks wrong. Everything is too big.||Please press the power-button to power Kilobaser off. Once the lights are out, immediately power-on again. Wait until the display looks fine. Alternatively please connect the display with an extra micro-USB cable (third cable) to an external USB-charger and reboot Kilobaser.|
Kilobaser can supply following accessories:
- Rubber sealing gasket
- Exhaust gas filter cartridge
- PCR vials
Taking the unit out of service
If the Kilobaser no longer works, it is to be appropriately disposed of.