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Aurora - Boreal Genomics

1. DNA load lt 40 ug Particulate content lt 200 mg non conducting particulates Solvent content Solvents are well tolerated if they do not disrupt the agarose gel or PMMA cartridge Samples that exceed these values are likely to produce outputs with low yield In addition DNA in the sample must be free to rapidly migrate in solution under electric fields and not bound to contaminants that will inhibit injection of DNA into the SCODA gel Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 Output The output of the Aurora system will have the following characteristics 40 60 ul depending on protocol Output buffer Same as gel buffer DNA yield gt 60 500 bp 50 kb 300 bp 50 kb in 0 25x TBE gels The Aurora will recover DNA fragments from the sample longer than 500 bp 300 bp in 0 25x TBE gels The presence of shorter fragments will not interfere with concentration but the shorter fragments may not be recovered Figure 21 illustrates the effect of fragment size on recovery schematically 100 500 bp 50 kb 50 0 ll 1 10 100 0 Yield DNA fragment length kb log scale Figure 21 SCODA DNA recovery by fragment length Contaminant rejection The Aurora platform has exceptional contaminant rejection capabilities Contaminant rejection was demonstrated using humic acids a complex mixture of organic molecules occurring naturally in soil Humic acids have a deep brown color and strongly inhibit PCR r
2. Contact plate mounting screws Figure 3 Contact plate drawer and features Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 10 Spring pins to connect to graphite electrodes in cartridge 7 aie 7 a Spring pins to connect Imaging window to high voltage electronics Figure 4 Contact plate Power button USB connection to Mains power input laptop and fuse Air exhaust Device firmware Ground ne update switch fault circuit e Ad GO interrupter Coolant connections Air intake Figure 5 Aurora rear panel Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 Front panel LEDs The Aurora front panel contains six LEDs arranged in the shape of a 1 sample cartridge that represent the electrical states of the six SCODA electrodes An additional center LED provides information about the status of the machine The electrode LEDs are white to indicate the electrode is at high voltage blue to indicate the electrode is grounded or off to indicate that the electrode is electrically disconnected The status LED in the center blinks white when the instrument is idle or paused is steady white when a run has been completed and is steady red when an error occurs during a run Each step of the SCODA process has a characteristic field pattern Figure 6 illustrates patterns associated with injection a focus b and a typical wash c Different protocols
3. The Aurora instrument requires a flat and level working surface capable of supporting the combined 35 kg 78 lb weight of the instrument laptop and chiller The Aurora instrument itself weighs 21 kg 46 lb the laptop weighs 1 5 kg 3 3 Ib and the chiller weighs 12 7 kg 28 lb Figure 7 illustrates the dimensions of the laptop instrument and chiller rene Figure 7 Dimensions of the Aurora and accessories Both the chiller and the Aurora instrument require extra clearance to allow for the free flow of air to their respective cooling systems as shown in Figure 8 The Aurora requires extra clearance behind the case to allow for the free flow of air into and out of the back panel while the chiller requires airflow into its left hand side and exhaust out of its right hand side Restricting this airflow can reduce performance and damage the instrument and chiller Warning Restricting airflow to the Aurora instrument or the chiller or allowing the ambient air temperature to rise above 25 C may cause damage to the Aurora instrument and chiller The chiller must be mounted at the same height as the instrument and care should be taken to ensure that the exhaust on the right hand side of the chiller does not mix with the air intake on the back panel Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 14 of the Aurora instrument If the chiller and Aurora are placed on the same surface the chiller must si
4. Contact Boreal Genomics for servicing Protocol uses imaging A camera is not connected Connect camera or disable imaging Error message Experiment folder not set Experiment will be saved to temporary folder Cause Current protocol contains invalid values User selected a cartridge that has more than one sample and did not select any samples to run Current protocol does not contain any blocks or has none of the existing blocks selected to run The gel boat drawer and possibly the contact plate drawer is open activating the safety interlock The instrument has not been calibrated properly prior to running The instrument hardware failure detection system was triggered and the instrument cannot be run in this mode This may indicate a serious hardware failure or it may possibly be a spurious error due to invalid run conditions not caught by the software Check RTEO8 for additional details No camera was detected although the selected protocol contains blocks that require imaging The experiment folder has not been selected by the user Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 Recommended solution Examine the protocol for any values that may be out of range Alternatively editing the faulty protocol in the Aurora software and then attempting to run or save the protocol will produce an error message indicating which values are invalid Select at least one sample to run or c
5. com Troubleshooting of protocol specific errors may be found in the documentation for each protocol Danger Under no circumstances should you attempt to resolve an error by servicing the instrument removing any of the exterior panels inserting foreign tools or objects into the machine or overriding the interlock systems Software error messages The error messages listed in this section are generated by the Aurora s fault handling software systems and are grouped into tables according to the screen at which they might occur Table 7 Pre run screen error messages Code Error message Cause Recommended solution PREO1 Aurora instrument not Aurora instrument is not Ensure that the USB cable from the found properly connected to the Aurora instrument is properly computer via the USB connected to the laptop communication cable Aurora instrument is not Power on the Aurora instrument powered on Other e Unplug re plug the Aurora USB connector into the laptop e Turn off the instrument for 5 Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 47 Code seconds before turning it back on PREO2 Error message Current protocol is not valid Current protocol uses 4 samples No samples are selected to run Current protocol has no blocks selected to run Gel boat drawer is open Instrument is not calibrated Contact Boreal Genomics to have your instrument calibrated Instrument Hardware Failure Detected
6. folders is done from the home screen seen in Figure 22 A list of folders that contain protocols is displayed on the left hand side To add a new folder click on the Add protocol folder EA button This will display the Add protocol folder pop up window seen in Figure 23 The Folder Alias field value is the value displayed in the Protocol Folder list It is available so the user can replace the folder path name which may be long with a shorter name Protocol Folders Protocols in C Boreal Genomics Protocols lt BOREAL Connection error No Aurora instrument detected Figure 22 Aurora home screen protocol management Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 42 For Folder Path field either type in a value or click on the Browse button to select a folder using the folder explorer When the correct values have been entered click Save Edit Protocol Folder 2 xj save Figure 23 Add protocol folder pop up To remove an existing folder from the Protocol Folder list select the folder and click the Remove Folder button ad To edit a current entry in the Protocol Folder list select the folder and click on the Edit Folder button a A prepopulated Add protocol folder window will be displayed and can be modified as necessary To create a new protocol go to the home screen of the control software Select a folder where
7. has entered DFU mode and the status bar will indicate the instrument has been disconnected At the same time Windows 7 will detect a new device connected to USB 2 Start ST DfuSe Demonstration software by going to Start gt All programs gt ST Microelectronics gt DfuSe gt DfuSe Demonstration You should now see the window shown in Figure 27 3 Check that in the top left Available DFU and compatible HID Devices section the drop down menu has the STM Device in DFU Mode selected 4 Select the DFU image to be uploaded by clicking the Choose button in the bottom right Upgrade or Verify Action section indicated Be sure to use the Choose button in the Upgrade or Verify Action panel and not the Upload Action panel Browse to the image received from Boreal Genomics representative the image should be formatted as a dfu file it is usually stored in C Boreal Genomics AuroraControl Slavelmage Aurora Slave dfu Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 62 a Dfuse Demo v3 0 0 Avallable DFU and compatible HID Devices STM Device in DFU Mode Supports Upload Supports Download Can Detach Enter DEU mode HIl0 detach Leave DFU mode Actions Select Target Internal Flash SFI Flash M25P64 NOR Flash Choose Upload Transtered data size O EB O Bytes of 0 EB O Bytes Time duration 00 00 00 Y O Manifestation tolerant O Accelerated Upload ST D
8. may use wash patterns that are different from pattern c which may appear to flicker rapidly Unless the SCODA software explicitly indicates an error condition this is normal behaviour and does not indicate a problem a Injection b Focus c Wash during focus Figure 6 Typical LED patterns Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 12 4 Installation and setup Read this section before unpacking and installing the Aurora After completing installation open the enclosed validation kit and follow the validation protocol to perform the validation run to ensure that the Aurora is functioning correctly Packing list e 1x Aurora instrument o 1x power cord o 1x USB cable e _1xlaptop o 1x power cord o 1x mouse e 2x coolant tubes 1 5 m length with fittings e 1x validation kit o Please see the validation protocol manual for a list of kit contents e 1x MSDS document package External chiller An external chiller is required to operate the Aurora As described in the Terms and Conditions of Sale for the Aurora instrument the warranty is only valid if the instrument is used with the following chiller e Vendor Solid State Cooling Systems e Part Number North America 120 VAC 10 400 2D 1 EF 90 e Part Number Europe 230 VAC 10 400 2D 2 EF 90 Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 13 Installation requirements Determining a location for the instrument
9. sample 5 1 channels 11 Block5 K 5 1 Wash Power Limit W 25 Interelectrode distance cm 3 88 4 Advanced Injection Threshold 20 Bias Inj ratio 3 00 JA Focus Saved on Mon Jun 6 16 00 59 2011 SW Ver 02 001R SW Build Jun 6 20 106 0001 AA D AURORA DNA CLEAN UP PROTOCOL DNA Clean Up Protocol for samples that have a conductivity less than or equal to 100 uS cm in 5 ml GENOMICS Connection error No Aurora instrument detected Figure 24 Aurora control software protocol edit screen To remove a block select the block to be removed and click on the Remove block button ie on The application will prompt for confirmation To change the execution order of blocks select the block that should be moved and click on either the Move block up button ie or Move block down button is to move the block up or down in the list To save a protocol click on the Save to disk button tai The application will prompt for the path where the protocol is to be saved Each block type has its own set of parameters At the top of each type of block the block duration is displayed in hours minutes and seconds Under the block duration field the other block parameters appear The following sections describe the parameter sets for each type of block Protocol wide settings Immediately after creating a new protocol the protocol wide parameters will be displayed You can return to the protocol w
10. the Aurora instrument labelled USB to the USB port on the laptop Powering the Aurora Plug the Aurora instrument into an AC power outlet using the provided power cord Ensure that the electrical circuit meets the requirements described in the Electrical requirements section on page 16 The receptacle for AC power is on the back panel of the Aurora instrument and is labelled either 120 VAC or 230 VAC Danger NEVER plug an Aurora instrument that is labeled on the back panel 120 VAC into a 230 VAC outlet or vice versa If you are unsure which voltage your Aurora has been configured for contact Boreal Genomics for support assistance before proceeding Next steps After installing the Aurora read the Operating the Aurora section to become familiar with the operation of the instrument Then use the enclosed validation kit and validation protocol to perform the validation run The validation protocol file and document can be found pre installed on the laptop in the folder C Boreal Genomics Protocols Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 21 5 Operating the Aurora This chapter will familiarize the user with the basic operation of the Aurora Read this section after completing the installation of the Aurora system and accessories described in the Installation and setup section beginning on page 13 and before beginning the validation run described in the enclosed validation run protocol manual R
11. 1 D Managing creating and editing protocols ss 42 Protocol Wide Setter A Ne ee 44 INJECTION DIOCK Setting Seien ao 45 FOCUS DIOCESANA e ee ph 45 AUTO MAPS SOTA SS AR ARA tab ent 46 Temperature Serna nadia 46 Es TTOUDI S AOOUNE it e 47 SO MWare error mess ares PR de td ee ns ip 47 Mechanical Ses usadas ad 53 Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 CR Ed a noe 54 Es Manten CS a SA M T Se 56 Preventative MAINTENANCE cccscccscccseccscccseccseccseecseececceecaeeceeceusceuscauscesecesceeceseceseceseesseetseenseetes 56 Removing and installing contact plates siennes 57 R pl cine the MAINS TUS RE ita dais 58 deining MEA ati aii oi 58 A SG nn ne 58 A Gen nn 59 G Upgrading the Aurora SoftWare it 60 Step 1 Uperadine Aurora PC SONWALE AS nr E A sc ne RAR nt en 60 Step 2 Device Firmware Update DEL SSSR aiii 61 Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 Table of Figures Figure Front view ot th Aurora did 9 Figure 2 Cartridge drawer and features sin he E 10 Figure 3 Contactiplate drawer and TE atesora 10 Figure 4 Contacto platero is en a 11 FISURE AUTO rear panels re A au Ha Ltd a de ele leds 11 Fieure o Typical LED PATTES AA A Hu tee AR 12 Figure 7 Dimensions of the Aurora and accessories sise 14 Figure 8 Clearances required for the Aurora and chiller coooooccnnonacnnnonanonnnonaconnnnaronnonanonnnonanos 15 Figure 9 Illustration of the
12. 3 BOREAL GENOMICS Aurora User Manual v2 30 2011 2012 Boreal Genomics Inc All rights reserved All trademarks are property of their owners http www borealgenomics com support borealgenomics com 302 2386 East Mall Vancouver BC V6T 173 1 604 822 4111 Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 Table of Contents a e e O PEO tien do a ct ce a 6 ZE INTO UCI D a Le 8 3 strument diagramo eio nanana E apices dan A Sas nt non none ni 9 FROME panel LEDS SSH LE RS A sd 12 INA TOR ana Stent 13 PAC ae dt ci 13 External co ler Le Re RS ni ee 13 Installation requirements nn NAS A A en 14 Unpacking and installing the AUrord cccccesseccccesscceceesececceeccceeecceeeusececeeneceeseesceeseenecesseeceeseeeeeten 17 Ds Operating INe AUTO a Ai 22 Preparine the AUO a a 22 Loading the Aurora Carte vise rit aia 25 Edad ties e o ni A 25 Winderstandine SEODA Protocol AN 27 Loading pre programmed protocols ccccoooccnncnocnnncnnncnnnnnaconnnnnocnnonanonnnonaronnonaronnonnnonnnonaronnonanionnos 27 DUNNE Uan aa 29 VA 16d OBL BS cB g PPC PO A gu A ee ee ee 30 EX EAC EINE UNG Salil A de 31 SHUTUNE COW EME IAS EME aisar DR nain een ad 32 APD NACE SAR SAR ne A SR T 33 As WUhnderstanaine SC ODA ai aa 34 B AUlrora concentration SDCCITICAUION iaa taaan den 38 MU a RE E AEA 38 OU O O O EE em ot 39 Contaminant teleco adas italia licita leida 39 C Suggestions for Upstream processes ini AA A AA 4
13. 32 hex key or driver remove and set aside the two flathead 8 32 screws that hold the contact plate in the contact plate drawer e Remove the contact plate from the contact plate drawer Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 57 Installing a contact plate e Open the cartridge drawer until it latches in its fully extended position e Open the contact plate drawer e Place the contact plate into the contact plate drawer frame so that the spring loaded contacts and circuit boards face down and to the back e Using a 3 32 hex key or driver insert the two flathead 8 32 screws that hold the contact plate in the contact plate drawer and tighten until secure Do not over tighten the screws to avoid cracking the plastic contact plate housing o Caution The screws should go in smoothly with minimal force Do not force the screws if they feel tight as they may not be inserted straight into their mating threads Close the contact plate drawer and cartridge drawer Replacing the mains fuse To replace the mains fuse on the power entry module use a screwdriver to open the fuse door and remove the red fuse holder from the power entry module The rated fuse is a medium blow 5x20mm 10A fuse LittleFuse part number 0234010 MXP If replacing the mains fuse does not resolve the problem contact Boreal Genomics for support Cleaning the Aurora Danger Disconnect the power supply before cleaning any of the electrical c
14. FU Mode Vendor ID 0483 Procuct ID 011 Version 0200 Procuct IO Version hz Available Sectors Double Click for more 256 sectors 128 sectors M2 26F 256 sectors Upgrade or Verify Action File Auyroraslawe diu endor ID 0485 Targets in file oo Auroras lave Procuct ID oona Version oona Verify after download Optimize Upgrade duration Remove same FF Upgrade File correctly loaded Abort Quit Figure 27 ST DfuSe main window Click Upgrade and then Yes when you are prompted to continue At the end of a successful I If at any point during the upgrade process the instrument disconnects from the 5 upgrade the progress bar at the bottom of the window will display Target 00 Upgrade successful computer please see Note 1 at the end of this section on how to proceed 6 Close the DfuSe Demonstration software and power cycle the Aurora instrument 7 Return to the Aurora Control software and the Aurora instrument should be detected automatically The status bar should display a message similar to Connected to instrument name Congratulations The upgrade is complete Note 1 If the device disconnected from the laptop while the firmware was updating the device firmware has been compromised and the device will not be able to connect to the laptop To resolve this issue turn off the instrument On the back panel of the instrument find the DFU switch While ho
15. Figure 10 Coolant connections on the chiller Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 18 Figure 11 External filter on the back of the chiller Insert the other ends of the fluid tubes with the white plastic fittings into either of the COOLANT WATER ports on the back panel of the Aurora instrument Press each in until the retaining ring snaps and then pull back to ensure it is secure It does not matter which tube is plugged into which coolant port on the Aurora Make sure the tubes are not strained or kinked at any point if necessary use zip ties or other fasteners to relieve strain on the tubes Take care not to pinch or restrict flow through the tubes Plug the chiller into an AC power outlet using the power cord included with the chiller following the directions in the Electrical requirements section above Filling the chiller The chiller s water reservoir cap is located at the top rear of the unit Fill the reservoir with tap water do not use deionized distilled reverse osmosis or other high purity water to just below the bottom of its neck Replace and tighten the water cap Flip the switch on the left side of the chiller to ON The chiller s internal pump will start and after a few seconds the display on the front of the chiller should display TANK LEVEL LOW as some of the fluid in the reservoir has now been pumped into the Aurora instrument Leave the chiller running for 1 minute to allow ai
16. Instrument not found The instrument has been disconnected Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 Recommended solution See RTEO9C Reconnect the instrument 52 Mechanical issues The errors described in this section are mechanical in nature and may not generate software error messages Indication Bubbling or gel deformation is observed in the gel during Aurora runs Table 10 Troubleshooting mechanical issues Cause The power dissipated in the gel exceeds the limits of the cartridge and protocol and excessive heating has caused the gel to melt or deform The bottom of the cartridge is not making effective thermal contact with the cold plate and the gel boils despite the fact that the power is within limits Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 Recommended solution Refer to protocol documentation for power and sample conductivity limits Ensure that samples are not more conductive than you expect and that you are using an appropriate cartridge for your sample Reduce injection and concentration fields until maximum power dissipation is within limits extending run time as necessary Carefully examine the bottom of the cartridge and the spreader plate for any debris that may produce gaps between the bottom of the cartridge and the surface of the cold plate Thoroughly clean the cold plate before repeating the run Ensure that 1 ml of water is placed on the col
17. al Genomics should be contacted as the instrument is either malfunctioning or has been used improperly To clear the error the instrument has to be power cycled However if no other measures are taken the error will likely re occur potentially resulting in a failed experiment The temperature controller cannot be controlled Contact Boreal Genomics for further troubleshooting steps The temperature controller cannot be controlled Contact Boreal Genomics for further troubleshooting steps The temperature controller cannot be controlled Contact Boreal Genomics for further troubleshooting steps The PS rail voltage cannot be set that is the set point PS voltage and the measured PS voltage differ by more than 50 Contact Boreal Genomics for further troubleshooting steps Device hardware is not compatible with the firmware Upload firmware compatible with existing hardware Contact Boreal Genomics for more details The states of the 5V and 24V interlock are not the same This indicates a serious hardware failure Please contact Boreal Genomics for assistance The instrument malfunctioned Contact Boreal Genomics for further troubleshooting steps Try diluting the conductivity of the sample to reduce the current If this fails to resolve the problem contact Boreal Genomics for further troubleshooting assistance 51 Code Error message Cause RTEO91 Power supply over current See causes for RTEO9O detected RTEOAO
18. ccurs with gt 100 ug of DNA for 5 ml cartridges e Molecules focus based on their value of k Shorter DNA molecules may not focus efficiently as k decreases for small DNA molecules e Contaminants with large k or those that bind DNA may focus along with the DNA Contaminant Rejection e Additional contaminant rejection can be obtained by applying a wash that is a DC field time multiplexed with the SCODA fields during focusing The SCODA fields constrain the DNA within the gel while contaminants are washed off the edge of the gel by the DC field Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 35 In general the stronger the wash the more quickly contaminants will be removed from the concentrated sample If the wash fields are too strong compared to the SCODA fields short fragments may be washed off the gel along with the contaminants because SCODA fields have larger effects on larger DNA molecules During a wash SCODA focus fields counterbalance the wash fields to retain DNA in the gel The focus fields are less effective at the lower field potentials required by more conductive gels so wash strength must be reduced and run times must be extended for samples too conductive for a 0 25x TBE gel Wash strength must be reduced to 15 or lower in 1x TBE Please see the documentation accompanying the cartridges and protocols for more information about wash times The Aurora will only remove contaminants that are not bound to th
19. ces The Aurora instrument is equipped with temperature control hardware that is capable of heating the cartridge to 60 C Use caution when opening the cartridge drawer and moving the cartridge to avoid burns from handling a hot cartridge touching a hot spreader plate or surrounding elements or splashing hot liquids Let the system cool down before handling any hot elements or use the temperature control system to cool the cartridge to a safe temperature after a run Pinch hazards The cartridge drawer and contact plate drawer present pinching hazards as the drawers are closing Always close the instrument drawers in a slow and steady motion taking care not to trap fingers or gloves in the instrument Chemical and biological waste safety The user is responsible for the safe use transport storage and disposal of any and all materials that may be considered a chemical or biological hazard Refer to accompanying material safety data sheets MSDS for safety information and handling instructions and comply with all federal state provincial municipal local and institutional requirements and guidelines for disposal Improper usage Usage of the Aurora instrument in a manner not specified by Boreal Genomics can prevent the proper operation of the protection provided by the instrument against shock and other hazards Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 2 Introduction The Boreal Genomics Aurora instrument uses Synchronou
20. contact Boreal Genomics Auto imaging settings The Auto Imaging settings are common to all blocks but the Wait block Table 6 Auto imaging settings Auto Imaging If the auto imaging checkbox is checked the instrument will automatically acquire images while the block is running If the checkbox is not checked no images will be acquired will be automatically acquired Sets the camera shutter speed Larger values will make images brighter ee brighter Images can be taken or saved directly from the auto imaging tab using the buttons surrounding the image preview window Snap Acquires an image The image can be used to decide if the shutter and gain values are appropriate The acquired image will not be saved see Save Save Saves the image in the preview window A pop up window will be displayed that allows the user to select the path of the saved file buttons Temperature settings All blocks also have a temperature setting This sets the temperature of the cold plate during the run Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 46 E Troubleshooting This section provides basic information on errors that the user may encounter while operating the Aurora In many cases you will be able to resolve the error by determining its cause and following the recommended solution as described below If these steps fail to resolve your error please contact Boreal Genomics support team support borealgenomics
21. correct relative placement of the Aurora and Chiller oo ommoooo 15 Figure 10 Coolant connections on the chiller sise 18 Figure 11 External filter onthe back Of UNG CHING vrs 19 Figure 12 Front panel and display of the chiller Temperature control is not active oooccccconocnnnmmoo 20 Figure 13 Front panel and display of the chiller with temperature control active 20 Figure 14 Aurora control software home SCFEEN ccccceseccecssscceceesececcesecceceesccecsesececseeceeseesceesausecetseneses 24 Figure 15 Cartridge loaded into the cartridge drawer Appearance of cartridge may differ please refer to protocol GOCUMENTATION A ia 26 Figure 16 Pipetting a sample into the sample chamber Appearance of cartridge may differ please refer toProtocol documenta sicario MR LAN esse c tt ie acte lc a ciel 26 FIBUTE 17 Pre run Screen nicotina ni tenta Ne diese tem otae Rcont Die 28 PICUPE Lo UNS CEN A dudit 29 Heure LO Posts creta da 31 Figure 20 Effect of sample conductivity on focusing time without additional contaminant rejection Contaminant rejection also increases run length in proportion to sample conductivity 37 Figure 21 SCODA DNA recovery by fragment length ss 39 Figure 22 Aurora home screen protocol management suisse 42 Fi ure23 Add brotocol older DOD UD ANS a ee Te te Nc en en 43 Figure 24 Aurora control software protocol edit screen sise 44 Figure 25 Contact pla
22. cs AuroraControl folder the previous software build is also located Navigate to the SW_bin folder of this older build and copy the AuroraProtoFolderLib pf file from there into the new build SW_bin folder overwrite any existing files 5 Return to the desktop and delete the archive file Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 60 6 Onthe desktop right click on the Aurora software shortcut and select Properties Update the Target text field so that it points to the Aurora executable in the new build folder Also update the Start in text field so that it points to the same new build folder 7 The first step is complete To check if it was performed successfully double click on the SCODA Control shortcut on the desktop The SCODA control application should start The title bar should report that you are running build yyyy mm dd hh mm MSVC and firmware VXX If the instrument is connected and turned on the software should report in the status bar that it is connected to an instrument If the status bar message indicates that the connected instrument runs the wrong firmware version you should proceed to step 2 If that is not the case you have finished the upgrade process Step 2 Device Firmware Update DFU This section provides information on how to upgrade the device firmware on the Aurora instrument Applying a DFU incorrectly may permanently damage the instrument Follow these instructions carefully to ensu
23. d plate to improve contact between the cartridge and cold plate 53 Chiller Indication Front display reads TANK LEVEL LOW Temperature on the front display does not reach the set point 20 0 C Front display reads RTD OPEN Front display reads FAN FAIL Front display reads PUMP FAIL or CHECK LINES Table 11 Troubleshooting chiller issues Cause The water level in the chiller has dropped below the minimum level The chiller is not operating The set point is incorrect The chiller has malfunctioned The temperature sensor in the chiller has failed or its connector has come loose Fan is supplying insufficient air to cool the chiller The liquid heat exchanger plate temperature is either too hot or too cold indicating pump failure Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 Recommended solution Turn off the chiller Unscrew the water reservoir cap that is located on the top rear of the instrument Top up the reservoir to just below the neck of the reservoir using tap water do not use deionized distilled reverse osmosis or other high purity water Replace the cap and screw down tightly Ensure that the START button has been pressed and the temperature controller is operating indicated by a plus or minus on the indicator LCD If the LCD displays a star this means that the temperature controller is in standby and should be started by pressing START STOP Pre
24. d to earth ground Do not attempt to remove any of the external panels Do not operate the instrument if panels have been removed or damaged or if fluid is ever observed leaking from the instrument Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 e The contact plate drawer and cartridge drawer are both designed with electrical safety interlocks that disable the high voltage elements of the system if either of the drawers is opened Do not attempt to bypass or disable the interlock systems To ensure that these systems operate as expected e All exterior panels must be in place and free of damage when operating the machine e The system must be plugged into a properly grounded outlet that supplies the correct voltage and current see the Electrical requirements section on page 16 To minimize the risk of electrical damage to the instrument and cartridge always pause the instrument before opening a drawer during operation Lifting moving and installing the instrument The Aurora instrument weighs 21 kg 46 lbs Use proper care and lifting techniques to avoid back strain and injury Install the system on a sturdy level surface capable of holding the instrument When lifting the Aurora support the instrument from the underside of the lower rounded white metal panels Do not attempt to lift the Aurora by the plastic front panel Ensure that the drawers are securely closed and all cables and tubing have been disconnected Hot surfa
25. dge into the Aurora e Check the bottom surface of the cartridge for any debris that may prevent it from sitting flat on the surface of the cold plate e Place the cartridge onto the surface of the cold plate Consult the protocol documentation to ensure that the cartridge is oriented correctly Ensure that the cartridge is sitting flat on the surface of the cold plate and sits within the registration features Loading the sample After preparing the cartridge transfer the sample into the center of the cartridge s sample well Figure 16 Different cartridges have different sample capacities and layouts Please consult the information that came with the protocol you are running for the recommended sample volume If the sample is smaller than recommended dilute it to the recommended volume in nuclease free deionized water or a very low conductivity buffer such as 0 01x TBE Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 25 Cartridge Cartridge registration features D Cartridge drawer Figure 15 Cartridge loaded into the cartridge drawer Appearance of cartridge may differ please refer to protocol documentation Figure 16 Pipetting a sample into the sample chamber Appearance of cartridge may differ please refer to protocol documentation Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 26 Close the cartridge drawer Resistance will be felt as the drawer closes apply gentle e
26. e DNA Contaminants bound to DNA are likely to focus and remain in the sample unless disassociated by heat or other denaturation prior to concentration High Molecular Weight DNA Due to the non mechanical nature of the SCODA process it is possible to concentrate high molecular weight DNA without shearing using a custom protocol Run times for HMW DNA can be 48 hours or more High molecular weight DNA gt 50 kb can become trapped in the gel at high SCODA fields and short periods SCODA fields and periods should be selected to minimize trapping In general the larger the DNA the longer the period and lower the fields that should be used A 4s period will concentrate up to 50 kb DNA at any field Increasing periods up to a maximum of 120 s may concentrate fragments up to 1 6 Mb at low fields High conductivity gels help reduce trapping of large fragments Lowering fields down to 8 V cm may assist in concentrating fragments up to 1 6 Mb For long periods DNA may move significant distances in the course of one period increasing the chance of losing DNA off the edge of the gel Final focus diameter will not be smaller than the path traced out by a single molecule during a full SCODA cycle Excessively long periods can increase the size of the SCODA focus creating losses outside the extraction well Run Speed The most important constraint on run speed on the Aurora platform is sample conductivity The conductivity of the sample determines w
27. eactions Because they carry a negative charge many DNA purification methods fail to partition them from DNA With electrophoretic washing and SCODA concentration it is possible to reduce unbound humic level concentrations to a level permitting PCR amplification without dilution in a 25 ul PCR reaction containing 1 ul of sample The sample used to demonstrate contaminant rejection contained 100 ng of pNEB206A 2 7 kb dsDNA and 500 ug humic acids Sigma H16752 in 5 ml 0 05x TBE A 25 ul qPCR reaction using ABI SYBR Green master mix containing 5 ul of the sample input was inhibited at dilutions lower than 1 1000 Concentration was performed using the DNA Clean Up protocol BG 106 0002 PCR sensitivity is expressed as the product of fold improvement in the lowest dilution that successfully amplifies because the Aurora s output is more concentrated than the input it could be successfully diluted further and still Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 39 amplify and fold improvement in the highest dilution that successfully amplifies because the Aurora s output is cleaner it amplified with less dilution Wash time 20 wash strength Increase in PCR sensitivity O h concentration only gt 10x concentration is DNA specific and improves DNA humic acid ratio 10x 50 000x PCR sensitivity reached a maximum at 2 h at which point the concentrated sample amplified normally without dilution Boreal Genomics Aurora Use
28. ead the safety instructions in the Safety information section beginning on page 6 before running the Aurora Preparing the Aurora Turn on the chiller by toggling the rocker switch on its left side The LCD display should read TEMP and a constantly updating temperature Press the START STOP button on the chiller s front panel to enable the temperature controller The icon on the left side of the chiller s LCD should change from a star to a plus or minus symbol to indicate that the temperature control is operating The temperature readout should update until it stabilizes at 20 0 2 C You do not need to wait for the temperature to stabilize before beginning a run Turn on the computer Log into the SCODA account using the password scoda all lowercase and start the Aurora control software by double clicking the shortcut labelled Aurora found on the Windows desktop Once the program home screen appears Figure 14 turn on the Aurora instrument by switching on the power switch on the back panel of the device Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 22 Aurora Operation Overview Turn on the Aurora and the chiller Start the chiller Make sure that a or page 22 appears on the chiller display to indicate that it is operating Prepare the cartridge and load the See protocol sample according to the directions for its manual accompanying protocol Open the cartridge drawer of the Aurora c
29. ee of kinks and blockages If this fails to resolve the problem contact Boreal Genomics for further troubleshooting steps See RTEO4O Decrease the electric fields and continue the run Decrease the electric fields and continue the run Check results of electrode contact test Contact Boreal Genomics Close the gel boat drawer Pause the run before opening the cartridge drawer If this message appears despite the drawer being closed ensure that the drawer is firmly closed and continue the run 50 Code Error message RTEO80 A serious hardware fault RTEO90 has been detected Please contact Boreal Genomics The power supply over current sensor was triggered This may be caused by 1 a salty sample 2 a current return path mismatch error Please check the sample saltiness is within range and retry If this error reoccurs please contact Boreal Genomics for assistance Cause Check the LCD panel for more details LCD can display the following messages RTEO81 TC Integral error RTEO82 Hardware heat sink over temp error RTEO83 TC current error RTEO84 HV PS voltage error RTEO85 Incompatible hardware ID RTEO86 Interlock switch error RTEOSFF Unknown error XX Caused by a sample that is too conductive salty Major GFI Internal short of electrode switches Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 Recommended solution For any of the messages below Bore
30. esent on the mains supply Power strips are acceptable provided they meet all listed electrical requirements Because the Aurora instrument laptop and chiller may together draw up to 12 5 A of current from a 120 V supply Boreal Genomics recommends that the Aurora instrument and supporting devices do not share an electrical circuit with other equipment to avoid triggering circuit breakers Table 2 contains more information about expected current draws Warning AN In order to ensure that the electrical safety features of the Aurora system operate correctly it is imperative that the grounding line of the power connection be intact and bonded to earth ground Danger NEVER plug an Aurora instrument that is labeled on the back panel 120 VAC into a 230 VAC outlet or vice versa If you are unsure which voltage your Aurora has been configured for contact Boreal Genomics for support assistance before proceeding Table 2 Electrical requirements Power draw W Current draw Current draw North America A Europe A Aurora instrument 450 S Aa 1 0 75 o 0 75 Chiller Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 16 Environmental considerations e The instrument is designed for operation in a laboratory environment temperature 15 to 30 C relative humidity below 60 and must not be operated in an environment where water may condense pool drip or splash onto orinto the instrument e The Aurora is rated
31. f water while actuating the pins by hand Allow the contact plate to dry before reinstalling it in the instrument If any spring pins are observed to be missing bent or corroded contact Boreal Genomics for assistance Check the tubing and fittings for leaks kinks and signs of wear Periodically examine all fittings and the entire length of tubing running between the Aurora instrument and the chiller Ensure that all tubes are free of wear damage and kinks and that there is no sign of leaking coolant or condensation Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 56 Check the instrument for sufficient airflow Sufficient airflow to both the chiller and the Aurora instrument is critical to ensure proper operation and the highest reliability of both systems Maintain a clear area around both systems free of clutter or any obstructions that would block free flow of air Removing and installing contact plates The Aurora instrument is designed to accommodate a wide range of cartridges New cartridges with different geometries can be used with the instrument simply by installing the corresponding contact plate Refer to Figure 25 while following the instructions below for removing and installing contact plates Contact plate mounting screws Figure 25 Contact plate mount Removing a contact plate e Open the cartridge drawer until it latches in its fully extended position e Open the contact plate drawer e Using a 3
32. for operation at elevations up to 2 000 m e The Aurora and chiller have cooling fans that produce 45 dBA 1 m of noise during operation e The chiller must be operated in an area with sufficient air circulation to allow the removal of 1100 W of waste heat while maintaining room temperature Unpacking and installing the Aurora Unpacking the Aurora Warning e At least two people are required to remove the Aurora instrument from its packaging materials Practice proper lifting techniques to avoid strain or injury e When moving the Aurora ensure the drawers are latched closed and support the Aurora only by the metal frame Do not grip the plastic panels e Oncethe Aurora instrument has been removed from its packaging it must sit upright on its feet Do not invert the Aurora Open the cardboard box for the main shipment Remove all items checking against the packing list on page 13 to ensure that all items were received and nothing was left in the packing material Lift the Aurora out of the box by its metal body Do not lift the Aurora by the plastic front panel Retain all packing materials until after the validation runs have been completed Place the instrument on an appropriate working surface as described in the Installation requirements section ensuring that there is 100 mm 4 in of space behind the instrument for airflow The instrument must be level and must not wobble To correct a tilt or wobble shim the instrument unde
33. for the run If no experiment folder is set the software will assign one by default in the system temporary folder and display a warning Using a separate folder for each experiment will help to keep images and data logs organized Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 28 Injection Running Fa p EANES O M Focus m _ Se BOREAL O 4 00 00 Injection Block Injec 10 EE 3000 OmC na lt BOREAL ENOMICS Connected to instrument Beta 25 Camera connected Yes Figure 18 Run screen Once all errors have been resolved the Run button will become enabled Click the Run button Lj to start the run The screen changes to the run screen Figure 18 To learn more about outstanding errors refer to Appendix E Troubleshooting on page 47 During a run The run screen allows the progress of the run to be monitored and controlled The run screen always displays information for both the 1 sample and 4 sample functionality When running 1 sample cartridges the fields related to samples 2 4 should be ignored To increase or decrease the amount of information displayed on this screen click on the add more details button ECS at the bottom of the screen Before the run starts the instrument checks that there is good electrical contact between the instrument and the cartridge and that the conductivities of the gel and sample are appropriate for an efficient injec
34. g it If it does not compress or does not return to its fully extended position after it is released contact Boreal Genomics for further troubleshooting and support guidance Check gel boat buffer reservoirs Refill with appropriate buffer as needed Contact Boreal Genomics Decrease the sample conductivity Contact Boreal Genomics Replace the gel boat with a new gel boat 49 Code Error message Cause 1 a leaking gel boat The conductivity of the sample 2 a sample that is too exceeds specifications causing conductive the power supply to exceed its 3 a major hardware fault current ratings Please check the gel boat A major hardware fault has for leaks and that the occurred sample conductivity is within range and retry If this error reoccurs please contact Boreal Genomics for assistance RTEO31 HV current return path Caused by a ground fault mismatch detected interrupt RTEO40 The cooling system The chiller is turned off and the temperature is out of range internal cooling system in the heat sink temperature Aurora is overheating X C Please check the An element of the cooling cooling water supply If this system Peltier element chiller error reoccurs please heat sink is not performing contact Boreal Genomics properly for assistance pop up dialog message RTEO41 Temperature out of range Caused by either the heat sink heat sink at X C or the spreader plate spreader plate at Y C temperature be
35. gel to drive DNA and all negatively charged molecules into the gel including any contaminants Then SCODA concentration fields are applied driving only the nucleic acids towards the centre of the gel Optionally Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 34 adding a wash field provides increased contaminant rejection compared to concentration alone Finally a concentration step is applied after the wash to ensure the DNA is collected in the centre of the gel The following are practical considerations when running the Aurora Injection e For efficient injection the sample when diluted to 5 ml must be less than one quarter of the conductivity of the SCODA gel to a maximum gel salinity of 1200 uS cm Samples of higher than one quarter the gel salinity will not inject efficiently and will result in poor recovery efficiency e Injection is based on total electric charge run through the sample and gel Each gel type has a fixed charge it can accept for the highest injection efficiency specified with the protocol assuming the one quarter salinity rule is followed This number will remain fixed for all samples that meet the gel salinity rule e Injecting greater than the recommended charge will result in short fragments over injecting and being lost off the edge of the gel Injecting less than the recommended charge may fail to admit all DNA in the sample chamber to the gel e Because charge based injection regulates the t
36. ges that have contained hazardous samples including many nucleic acid stains may be considered hazardous in your jurisdiction Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 31 Shutting down the instrument To shut down the Aurora instrument first use the software to stop or pause any run that may be in progress Flip the power switch on the rear panel to the off position and then flip the rocker switch on the left side of the chiller to the off position The laptop may also be shut down Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 32 Appendices Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 33 A Understanding SCODA SCODA Synchronous Coefficient of Drag Alternation is a nucleic acid separation concentration and purification method based on electrophoretic motion of molecules in a gel It is fundamentally different from both DC and AC electrophoretic methods such as pulsed field gel electrophoresis in that all conventional electrophoretic separations tend to be dispersive with bands increasing in breadth through the duration of a run SCODA on the other hand employs a novel physical parameter k associated with molecular motion of long charged molecules in a gel to specifically concentrate all nucleic acids from a sample to a single location This concentration makes SCODA inherently non dispersive in the sense that running SCODA longer within reason does not degrade its focusing pro
37. hange the cartridge to a one sample cartridge in this case the one sample is selected by default Edit the protocol and select at least one block to run Close the gel boat drawer Contact Boreal Genomics to have your instrument calibrated This procedure should only be performed by Boreal Genomics technicians The hardware failure detection system can be reset by power cycling the instrument If the hardware failure error persists contact Boreal Genomics for further troubleshooting steps Ensure that a camera is connected to the instrument Power cycling the instrument can restore camera connectivity Table 8 Pre run screen warning messages Recommended solution Select an experiment folder 48 Code Table 9 Run screen error messages Recommended solution Error message Failed electrode contact test on X reading YV instead of ZV Check electrode contact and fill all buffer chambers Additional details RTEO10 Electrode contact test malfunction Additional details Failed injection conductivity test Sample conductivity is too high Injection might fail Additional details Injection conductivity test malfunction Additional details A high voltage current short was detected This may be caused by RTEO11 RTEO20 RTEO21 RTEO30 Cause The electrical contact test between the instrument and the gel boat has failed This can result because one or more of the following occu
38. hich cartridge can be used since SCODA gels must be about 4 times as conductive as the sample for complete injection It is advantageous to use the least conductive gel type possible as more conductive gels must be run at lower fields to prevent overheating and take longer to run Always choose the lowest gel conductivity that the sample will permit Figure 20 illustrates example run times for focusing without wash as a function of sample conductivity Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 36 j 1x TBE gel 0 5x TBE gel 0 25x TBE gel Focusing time h N 0 100 200 300 Sample conductivity uS cm Figure 20 Effect of sample conductivity on focusing time without additional contaminant rejection Contaminant rejection also increases run length in proportion to sample conductivity Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 37 B Aurora concentration specification This appendix describes the Aurora system s capabilities In some cases these values represent conservative estimates If you have questions about these values please contact our technical support team at support borealgenomics com Input The table below is intended to describe the types of samples that the Aurora is capable of handling Please see documentation for individual protocols for more information lt 200uS cm DNA fragment size lt 50 kb dsDNA longer than 50 kb but up to 1 Mb will require longer run times
39. ide parameters later by clicking on the Parameter wide settings button ty at the top of the block list Table 3 provides a brief description of the protocol wide parameters available Table 3 Protocol wide settings Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 44 Cartridge Type The cartridge type used for this protocol Depending on cartridge selection the imaging FOV will automatically be resized and rotated to display the gel area with the sample chamber on the right The 4 sample cartridge choice is currently not supported Injection Threshold If the voltage drop across the gel during injection exceeds this value a warning will be displayed Appropriate values for this parameter will ensure that the DNA is transferred efficiently from the sample chamber into the gel Power Limit W If the power dissipated in the gel during a run exceeds this value a warning will be displayed Appropriate values for this parameter will ensure that the gel will not melt during the SCODA run Protocol Description This text is displayed when the protocol is selected on the home screen Experiment Comment This text will be displayed automatically in the Experiment Comments Template text field in the Pre Run screen see Figure 17 Injection block settings Table 4 Injection block parameters Parameter Name Description Injection Voltage Specifies the voltage to be applied across the sample chamber and SCODA gel during injectio
40. ing outside their event log range RTEO50 Power limit for sample X Average power has exceeded exceeded Average power the maximum threshold and the Y W Power limit Z W Run instrument has paused the run Paused in order to avoid boiling the gel RTEO61 Current limit exceeded Average current has exceeded Current X mA Current the maximum threshold limit Y mA Run Paused RTEO62 Sample X current is below The current measured for one of minimum current limit the samples is below the Sample X current Y mA minimum threshold current Current limit Z mA Run Paused RTEO7O Cannot resume run The gel boat drawer and Cartridge drawer is open possibly the contact plate pop up dialog message drawer is open activating the safety interlock RTEO71 Cartridge drawer open The cartridge drawer has been WARNING Opening the opened without the run being cartridge drawer without paused pausing the run can result in permanent damage to the instrument Please pause the run before opening the cartridge drawer pop up dialog Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 Recommended solution Dilute the sample to an appropriate conductivity level or drop the voltage of the PS Contact Boreal Genomics for further troubleshooting steps if unable to resolve this issue See error RTEO30 Turn on the chiller Wait until the heat sink temperature drops below 35 C and continue the run Ensure that all tubes are fr
41. instructions Turn the chiller and instrument off immediately and contact Boreal Genomics for instructions 55 F Maintenance Preventative maintenance The Aurora system requires very little maintenance if it is used within its specifications The following preventative maintenance tasks can be performed periodically in order to ensure consistent and reliable operation of the system and to reduce the likelihood of errors Check the contact plate for salt buildup and signs of wear Each contact plate contains a number of spring loaded contacts that provide electrical connection between the contact plate and the cartridge and between the contact plate and the high voltage driving electronics inside the instrument These spring pins are cycled every time the cartridge drawer or contact plate drawer is opened and closed and may be subjected to considerable wear Furthermore there is a possibility that buffer from the cartridge may splash up onto the underside of the contact plate Over time the accumulation of buffer on the spring pins may leave salt deposits that interfere with the motion of the spring pins Periodically remove the contact plate from the contact plate drawer refer to the Removing and installing contact plates section below and closely examine the surface of the printed circuit boards and the spring pins for signs of wear or debris If salt deposits are observed on a spring pin rinse it several times with a small amount o
42. lay will switch back to the current temperature as in Figure 12 note that the star symbol on the far left hand side indicates that the temperature control is not yet enabled 2 Press the START STOP key to activate temperature control The icon at the far left of the display will change from a star to a plus or minus symbol to indicate that the chiller is active The coolant temperature should eventually stabilize at 20 0 2 C as shown in Figure 13 Figure 13 Front panel and display of the chiller with temperature control active Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 20 The set temperature will persist in memory if the chiller is turned off Once it has been set once to 20 C it should never need to be reset Every time the chiller is powered on with the main switch however the START STOP key will have to be pressed to turn temperature control on Setting up the laptop Place the laptop on the working surface next to or on top of the instrument Plug the laptop into an AC power outlet using the AC adapter provided with the laptop following the directions in the Electrical requirements section above Plug the mouse into any uncovered USB port on the laptop note that for certain laptop models Boreal Genomics will install plastic covers on certain USB ports to prevent using them with the Aurora due to driver incompatibility use only uncovered ports Use the provided cable to connect the port on the back of
43. lding down the DFU switch turn on the instrument the instrument will be detected as a DFU device Continue the DFU procedure from step 2 Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 63
44. lean the cold plate place 1 ml of water on the center of the cold plate to pages 25 6 improve thermal contact and place the cartridge firmly on the cold plate Start the Aurora Control software and select the protocol you wish to run Press page 27 Run MS Defi i t fold d oe an experiment folder and press Dage 28 Once the run has ended extract the sample according to the protocol directions JEE See protocol manual Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 23 Protocol folder list Protocol list in System Protocol Folders Protocols in System C Boreal Genomics Protocols Protocol Description Status bar xs BOREAL Connection error No Aurora instrument detected Figure 14 Aurora control software home screen The control software will automatically connect to the instrument Check that e the status bar message indicates that an instrument is connected and displays the name of the instrument e the status bar message indicates that a camera is connected and e the front display of the Aurora instrument displays the message Idle If the camera or Aurora does not connect successfully unplug the laptop end of the USB cable connecting the Aurora to the laptop wait five seconds and reinsert the cable Repeat as necessary A note about temperature control The SCODA process can generate a significant amount of heat in the small volume of the agarose SCODA gel Contr
45. n Injection Type Determines whether injection will run for a set time time limited or until the integrated amount of current through the sample reaches a threshold charge limited If the Charge Limited injection type is specified the block duration field is replaced by an injection charge field Injection Warning Enabling this check causes the Aurora to perform an injection conductivity test before the block runs The conductivity test is used to assess if the gel versus sample voltage drop ratio is appropriate for an efficient injection The software will issue a warning if this ratio is above the predefined threshold value specified in Protocol Wide Settings Focus block settings Table 5 Focus block parameters SCODA Field Specifies the electrical field strength to be used in the focus block seconds 4 s periods are standard for most SCODA runs field pattern Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 Wash Strength Sets the duty cycle of the wash field with respect to the SCODA field Using high wash strength values can result in the DNA sample being washed off the gel Minimum current warning Monitor the magnitude of the SCODA current and warn the user if the value drops below a minimum threshold of 3mA This is used to detect possible failures of the electrical contact between the cartridge and the contact plate To obtain a description for the advanced block parameters please
46. nd images acquired during an experiment are saved and e add any experiment specific comments which will appear in the experiment log files and in the captions of any acquired images Before starting a run review and correct any error conditions by following software prompts Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 27 Protocol Details Outstanding Issues II Experiment Folder C Boreal Genomics Experiment ExpA4224 Run 2011 05 12A a Experiment Comments Experimenter name John Matrix Sample 42A Run 1 3 lt BOREAL GENOMICS Connected to instrument Beta 25 Camera connected Yes Figure 17 Pre run screen Understanding experiment folders Before starting a protocol the Aurora control software will prompt for an experiment folder at the Pre Run screen During a run the Aurora logs information about the progress of the run to files in the experiment folder These include EventLog html which contains a summary of events during the run and the time they occurred and DataLog csv which contains debugging and diagnostic information The experiment folder is also where any automatically captured images are saved Though the Aurora software is designed to avoid overwriting data Boreal Genomics recommends creating a new experiment folder for each sample To select an experiment folder click the Browse button Lr and in the window that pops up choose or create an experiment folder
47. ol of the gel temperature is critical to maintain optimal performance The Aurora cartridge rests on an actively cooled cold plate to draw heat away from the gel In order to transfer heat from the cartridge to the cold plate it is critical that the cold plate and bottom of the cartridge are entirely clear of any debris that might create gaps between the two surfaces Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 24 Loading the Aurora cartridge Important Take care not to spill buffer samples or other materials inside the Aurora Clean any spills before closing the cartridge drawer Consult the protocol documentation for specific advice on loading the cartridge you plan to use into the Aurora In general to load a cartridge into the Aurora instrument e Prepare the cartridge according to the documentation accompanying the protocol you are following e Inspect the cartridge for any signs of leaking buffer bubbles in the gel or damage to the graphite electrodes If you notice damage contact Boreal Genomics e Gently pull the cartridge drawer out of the instrument until it latches in the fully opened position e Check the cold plate for debris that may prevent the cartridge from sitting flat e Pipette 1 ml of water onto the center of the cold plate to improve transfer of heat from the cartridge to the cold plate Important Ensure that the cold plate and the bottom of the cartridge are clean before loading the cartri
48. ontacts Do not attempt to reach into the cartridge drawer to clean inside the Aurora instrument Clean the cold plate regularly with water to prevent the accumulation of debris or salt precipitates that could interfere with the thermal contact between the cold plate and cartridge The casing of the Aurora can be cleaned as necessary with a mild soap solution Avoid organic solvents that may damage the plastic housing of the Aurora instrument Chiller Perform the following preventative maintenance steps every 2 months e Perform a pull test on the fluid tubing running between the chiller and the Aurora Instrument Ensure that the tubing is securely plugged into the chiller and instrument To perform a pull test firmly grasp each fluid line and pull straight outwards with moderate force No tubing should come loose Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 58 e Check for leaks from the chiller particularly from connection points for tubing No coolant should leak out at any time If any coolant is observed to leak turn the chiller and instrument off immediately and contact Boreal Genomics for troubleshooting information Instrument airflow The airflow into and out of the Aurora and the chiller must be maintained in order to ensure that the systems do not exceed their operating temperature and are able to dissipate the required heat to the environment Periodically check the layout of the Aurora and chiller
49. otal electric field seen by the gel it is more reproducible than specifying a fixed injection time As a result charged based injection is used by default on all Aurora systems Concentration e SCODA concentration on its own provides up to 100 fold contaminant rejection due to DNA concentration which increases the ratio of DNA to unbound contaminants e The lower the gel conductivity the faster the SCODA wash and concentration will be High conductivity gels i e 1x TBE result in very long run times overnight with wash and should only be used for high conductivity samples that cannot be diluted e The SCODA concentration duration scales with the inverse of the square of the electric field If the SCODA fields are reduced by half run time will increase by a factor of four e Heat dissipation from the gel is the limiting factor in the speed of the SCODA concentration The heat produced in the gel is proportional to the conductivity of the gel and to the square of the applied electric field If the conductivity is doubled heat will double If the fields are doubled heat production will go up by a factor of four e Each protocol has a maximum power dissipation it can sustain during concentration specified with the protocol before the gel will fail bubble or melt and sample will be lost e Excessive focused DNA mass can perturb the electric field geometry and lead to unusual focus locations or shapes and poor recovery Typically this o
50. perties and DNA from multiple samples may be overlapped onto a single SCODA focus The SCODA concept is to generate periodic motion of DNA using electric fields while synchronously altering the mobility or drag coefficient of the molecule with a second electric field to cause a net drift and subsequent concentration Because concentration results from the non linear velocity response of the molecule to increasing electric fields k and appears to be unique to long polymers such as DNA this allows SCODA to efficiently select only nucleic acids for concentration In practice this drives nucleic acids toward a buffer well in the centre of the SCODA gel to a location free of electrodes More of the physics behind SCODA concentration can be found in the Proceedings of the National Academy of Sciences 2009 Sep 1 106 35 14796 801 The main advantages of SCODA arise because concentration based on k is unique to nucleic acids This enables efficient recovery of DNA from up to 5 ml samples while partitioning contaminants resulting in excellent contaminant rejection It also allows for high recovery efficiency even down to small numbers of DNA molecules In addition it provides a 100 1 volume reduction from a single sample as the output DNA is delivered in 50 ul of buffer The main elements of SCODA purification are injection contaminant rejection or wash and concentration During injection an electric field is applied across the sample and SCODA
51. r Manual BG 2002 07 004 v2 30 40 C Suggestions for upstream processes In general any lysis or preparative method that produces mobile DNA in solution with conductivity lower than 100 uS cm when diluted to 5 ml can be run directly in SCODA in a 0 25x TBE gel Some examples include standard phenol chloroform preparations silica based methods and commercially available DNA preparation kits Larger volumes or higher salinity samples can be run in SCODA by dilution and running multiple injection and focusing blocks as long as the conductivity of each 5 ml sample is below 100 uS cm Higher conductivity samples may be run as a single sample but will yield less DNA Higher salinity gel cartridges can increase the conductivity limit to 300 uS cm in exchange for longer run times Because the Aurora can handle sample volumes up to 5 ml it may be possible to improve yields of upstream processes by eluting in larger than usual volumes since DNA will be re concentrated by SCODA It may also be possible to eliminate potentially lossy washes or other contaminant rejection steps from upstream processes as SCODA concentration will provide contaminant rejection Finally the application of heat or other denaturants immediately prior to SCODA may help remove contaminants that are initially bound to the DNA Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 41 D Managing creating and editing protocols The maintenance of protocol files and
52. r bubbles to escape then turn the chiller off and top up the reservoir with tap water again to just below the bottom of its neck Replace and tighten the water cap Note that it is important to only fill the tank while the chiller is off filling the tank while the chiller and pump is running will overfill the tank and result in tank overflow when the chiller is switched off Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 19 Before proceeding allow the chiller to run for at least 4 hours to allow all air bubbles to escape Repeat the filling steps described above until the reservoir remains full and the chiller does not display the TANK LEVEL LOW error instead it should display the word TEMP and the temperature of the coolant in degrees Celsius The chiller has now been successfully filled Programming the chiller After turning the chiller on the chiller should display a star the word TEMP and a constantly updating measurement of the temperature of the circulating coolant The chiller has four input keys UP DOWN ENTER and START STOP The front panel and display are shown in Figure 12 Note that the star icon on the far left indicates that temperature control is not active Figure 12 Front panel and display of the chiller Temperature control is not active To set the chiller temperature 1 Press the UP or DOWN keys to change the set temperature to read 20 0 C Press Enter to set the temperature The disp
53. re a successful upgrade Prerequisites To perform a DFU obtain the firmware image to be deployed on the instrument If you have just upgraded the Aurora control software it is typically found at C Boreal Genomics AuroraControl Slavelmage Aurora Slave dfu You will also need the ST Microelectronics DfuSe software which is installed on all laptops delivered with Aurora instruments DFU procedure Power on the Aurora instrument and start the Aurora control software The control software should detect the instrument connected check that the status bar in main window displays the message Aurora detected but is running the wrong software version as in Figure 26 If the software shows that an Aurora is connected normally do not proceed the upgrade process is complete Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 61 _ AURORA E Protocol Folders Protocols in C Boreal Genomics Protocols DNA Clean up Protocols Kej s erg E Update Device Firmware Validation protocols Calibration Mode About DNA Clean up protocols LA 2 4 Protocol Description pee E lt gt BOREAL Connection error Aurora is running the wrong firmware version au4s002 Compatible firmware au4s001 FE Figure 26 Aurora control DFU mode selection 1 From the home screen select System and Update Device Firmware highlighted in Figure 26 Enter scoda123 as password A pop up window will announce that the device
54. rneath one or more feet as needed Remove all packing tape and material from the Aurora instrument including the foam from inside the cartridge drawer Placing and connecting the chiller Place the chiller on a sturdy stable working surface to the right of the instrument Ensure that there is at least 80 mm 3 in of clearance on both the left and right side of the chiller for air flow Locate the push to connect fitting on the chiller labelled PROCESS IN see Figure 10 If there is a solid plug in the fitting remove it by pushing the outside collar of the fitting down with one hand and simultaneously pulling straight backwards on the plug with the other hand The plug should come out Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 17 with minimal force if the collar is fully depressed Once the plug has been removed or if there is no plug provided insert the bare end of one of the provided fluid tubes into the fitting Push the tube in until it stops and then pull back to ensure it is secure The chiller will come with a pre installed tube running from the port labelled PROCESS OUT to the port on the blue external filter on the back of the chiller shown in Figure 11 labelled IN Insert the other provided fluid tube into the port on the external filter labelled OUT again pressing until it stops and then pulling back to check that it is secure Follow the same procedure to remove a plug for this port if a plug is present
55. rred Gel boat improperly seated A gel boat type other than the one specified in the protocol details is used The graphite electrodes in the gel boat have been damaged and do not make contact with the spring loaded pins on the contact plate Contact plate electrode pins are damaged The buffer in one of the buffer reservoirs is depleted A hardware failure occurred during the electrode contact test The conductivity of the sample is too high A hardware failure occurred during the injection conductivity test The gel boat is leaking and currents from the gel boat are leaking into the cold plate Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 Ensure the gel boat is properly seated on the top of the cold plate Replace the gel boat with a gel boat that is compatible with the protocol and contact plate that are being used Carefully examine the gel boat for signs of wear to the graphite electrodes If damage is observed replace the gel boat Remove the contact plate refer to Appendix F Maintenance and ensure that no spring pins are damaged missing or stuck in a compressed position If a spring pin is stuck in the compressed position examine it closely for any sign of debris especially salt crystals that may be interfering with its motion Remove any salt from the spring pins by rinsing with deionized water If there is no salt or debris try compressing the spring pin by hand and releasin
56. s Coefficient of Drag Alteration SCODA electrophoretic DNA manipulation technology to concentrate and purify DNA from challenging samples Instead of separating DNA based on its chemical properties or by affinity SCODA technology purifies DNA based on its physical properties selecting for long charged polymers As a result the Aurora instrument provides exceptional contaminant rejection over a broad range of contaminants In addition the Aurora delivers up to 100 fold concentration of DNA from a single sample from several hundred molecules up to 100 ug of DNA The Aurora accepts up to 5 ml of low conductivity lysate and purifies the DNA into a final buffer volume of up to 60 ul To ensure efficient DNA injection and recovery samples must have a conductivity not exceeding 200 uS cm in 5 ml More details regarding specific sample processing upstream of the Aurora are provided as separate protocols by Boreal Genomics Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 3 Instrument diagrams Instrument text display Front panel LEDs Contact plate drawer Cartridge drawer OD a gt D Figure 1 Front view of the Aurora Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 Cartridge drawer cover Cold plate Cartridge registration features Cartridge drawer Figure 2 Cartridge drawer and features Contact plate Contact plate drawer
57. ss the UP or DOWN keys to change the setpoint to 20 0 C Inform Boreal Genomics of the problem and contact Solid State Cooling Systems for troubleshooting information Inform Boreal Genomics of the problem and contact Solid State Cooling systems for repair instructions Power the chiller off to reset the alarm Check that the side air inlet and outlet gratings are not blocked at least 3 inches of clearance on either side are required If airflow is not blocked inform Boreal Genomics of the problem and contact Solid State Cooling systems for repair instructions Power the chiller off to reset the alarm Check that the coolant connectors are firmly seated and 54 or a blockage in external plumbing lines Front display is blank no The chiller is not powered display The chiller has malfunctioned The chiller is leaking coolant Tube fittings are loose Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 that there are no kinks in the tubing running from the chiller to the Aurora instrument or from the chiller to the external filter on the back of the chiller If there are no kinks contact Boreal Genomics for further troubleshooting instructions Check the electrical connection to the chiller and the electrical outlet it is plugged in to Ensure that the rocker switch on the left side of the chiller is set to ON Inform Boreal Genomics of the problem and contact Solid State Cooling systems for repair
58. t to the right of the Aurora as shown in Figure 9 The chiller is connected to the instrument by two tubes of length 1 5 m 5 ft The laptop must be connected to the instrument in order to control it The laptop can be placed anywhere within the 1 m 3 ft reach of the provided USB cable typically on the working surface next to the instrument or on top of the instrument q ae behind for air flow y 80 Mc air flow 80 mm on side for ee flow Figure 8 Clearances required for the Aurora and chiller Figure 9 Illustration of the correct relative placement of the Aurora and chiller Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 15 Electrical requirements Table 1 Aurora electrical supply requirements and model compatibility Voltage Compatible Aurora model number s listed on back panel North America 120 VAC 60 Hz AURO1A NA AURO2A NA AURO3A NA AURO3B NA AURO3C NA AURO3D NA AURO3E NA 230 VAC 50Hz AURO2A EU AURO3B EU AURO3C EU AURO3D EU AURO3E EU For North American locations three standard North American AC power receptacles 120 VAC 60 Hz three prong grounded NEMA 5 15 are required For European locations three standard European AC power receptacles 230 VAC 50 Hz two pronged grounded CEE 7 4 are required Voltage fluctuations must be limited to 10 of the of the nominal voltage and transient overvoltages must be limited to typically pr
59. te MOUN ES a Te D dr 57 Figure 26 Aurora control DFU mode selection cocomoonononnoronnonoricnonaciononaoronconoricnonariononaaronconaracnonasos 62 Figdre 27 ST D OSe MA WINdO Was A M ie nn tent eee re td 63 Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 1 Safety information The SCODA concentration technology applied by the Aurora instrument is driven by high voltage electric fields Improper use of the instrument may expose the user to electrical hazards The Aurora has safeguards designed to protect the user from these hazards Please read the information below on these safety features before using the instrument Danger Never attempt to service an Aurora system or to remove any part of the enclosure unless instructed by the user manual or Boreal Genomics staff Do not insert any tools or objects into the machine other than those required for regular instrument operation Certification and standards information The Aurora instrument has been certified for safety compliance with the harmonized CSA UL and EU standard IEC 61010 1 Safety requirements for electrical equipment for measurement control and laboratory use Electrical safety The Aurora system is designed with safety features to prevent the user from accessing hazardous parts of the system particularly the high voltage electronics e All hazardous electrical elements of the system are encased within a metal and plastic enclosure that is bonde
60. the new protocol will be created and click on the New Protocol button ME If a pre existing protocol is to be modified select the protocol of interest and click the Edit Protocol button MEA In either case the edit screen will be displayed as in Figure 24 You can edit protocol wide parameters and add remove and edit blocks in the edit protocol screen SCODA protocols are composed as sequences of blocks Each type of block performs a unique function There are four types of blocks e Injection block Draws DNA into the SCODA gel from the sample chamber e Focus block Concentrates DNA in the SCODA gel into the extraction well Focus blocks can also have an electrophoretic wash superimposed for added contaminant rejection e Wait block Pauses for a set time or waits for user intervention e Advanced block Runs custom field patterns and operating conditions Newly created protocols do not yet contain any blocks To add a block click on the Add block button ie under the Protocol Blocks list field A drop down menu listing the types of blocks that may be added will pop up Select one of the block types to add it To change the default name of a newly created block double click on the block name in the protocol list and rename the block Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 43 SampleProtocol 4 02 00 BRA or bh Protocol Wide Settings ra Injection Cartridge Type 1 sample 5mm height 5 ml
61. the software to the home screen The most recently run protocol can be edited by clicking on the Edit Protocol button The current protocol can be saved by clicking on the Save button tay or re run by clicking on the Re Run button Additionally the user can add a post run comment to the event log indicating any special events that occurred during or after ther run Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 30 Event Log 200 00 lt BOREAL GENOMICS Connected to instrument Beta 25 Camera connected Yes Figure 19 Post run screen Extracting the sample Use caution when opening the cartridge drawer and removing the cartridge Test the temperature of the cartridge before picking it up to avoid burns Follow the directions in the protocol manual to extract concentrated DNA from the Aurora cartridge The expected output volume will depend on the type of cartridge being used and may vary slightly This variability should not affect the performance of the Aurora Concentrated nucleic acids produced by the Aurora are suitable for quantification by quantitative PCR and other standard methods Nucleic acids may be visualized after concentration by DC gel electrophoresis in the presence of a suitable dye Dispose of the cartridge and contents following all applicable policies laws and regulations Aurora cartridges buffers and gels are non hazardous as shipped though cartrid
62. tion The front panel LEDs will flash during the check If the instrument detects a problem the Aurora software issues an error See Appendix E Troubleshooting for more information about error conditions and advice for resolving them Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 29 The Aurora waits until the cold plate reaches its set temperature before beginning the run which typically takes 1 2 minutes The Aurora LCD will display Starting Block and the software will display the message Setting Temperature During the run the control software proceeds through each block in the block list in sequence While the run is proceeding the instrument LCD panel displays the message Running and the time remaining in the run Because the progress of injection is measured by the total amount of charge that has passed through the sample and not by time the displayed total time will not include the injection block and the timer will not count down while the sample is injecting The countdown will begin after injection finishes If the instrument requires user input the central LED on the front panel blinks white If the instrument encounters an error during a run the central LED turns steady red and the Aurora control software displays a pop up window with information pertaining to the error If imaging is enabled photographs of the cartridge will appear as they are captured Whether imaging is enabled or not you can manuall
63. to ensure that there is adequate Spacing on all sides that the area has not been cluttered and that fan and vent holes have not been obstructed Refer to the section on Determining a location for the instrument starting on page 14 for detailed information on adequate airflow Boreal Genomics Aurora User Manual BG 2002 07 004 v2 30 59 G Upgrading the Aurora software This section provides instructions for upgrading the Aurora control software The upgrade process has two steps Step 1 upgrades the software on the laptop Step 2 upgrades the software also known as firmware on the actual instrument Please finish step 1 before starting step 2 Step 1 Upgrading Aurora PC software This section provides information on how to upgrade the Aurora control software on the laptop computer accompanying the Aurora instrument Prerequisites Archive file containing two folders Bin and Slave received from Boreal Genomics Upgrade procedure 1 Copythe ZIP update archive to the laptop of the Aurora system that needs to be upgraded 2 Unzip the archive file on the desktop You should now have a folder with a name of the form vyyy mm dd rXXX indicating the update build release date and build release number on the Windows desktop 3 Move the folder that was just unzipped from the desktop to C Boreal Genomics AuroraControl In the same C Boreal Genomics AuroraControl folder the previous build folder is stored 4 Inthe C Boreal Genomi
64. ven pressure to both left and right sides of the drawer until it is secure Avoid quick movements that will cause the liquid in the cartridge to splash Understanding SCODA protocols SCODA protocols are composed as sequences of blocks There are four types of blocks each of which performs a unique function e Injection block Draws DNA into the SCODA gel from the sample chamber e Focus block Concentrates DNA in the SCODA gel into the extraction well Focus blocks can also have an electrophoretic wash superimposed for added contaminant rejection e Wait block Pauses for a set time or waits for user intervention e Advanced block Runs custom field patterns and operating conditions Refer to Appendix D Managing creating and editing protocols for more detailed information on blocks and their parameters Loading pre programmed protocols From the home screen previously saved protocols can be loaded or new protocols can be created To run a pre programmed protocol such as the validation run or other protocols supplied by Boreal Genomics e Select the folder where the protocol resides in the left hand side of the screen e Select the protocol to be run in the right side of the screen and click The control software will advance to the pre run screen The pre run screen Figure 17 allows the user to e review the protocol to be run e set the path for the experiment folder where experiment log files event log and data log a
65. y capture an image by pressing the camera button 8 Manually acquired images will not be saved unless you press the save button El When all blocks in the protocol finish running the control software displays the post run screen the instrument s LCD screen displays the message Run Done and the center LED glows steady white The run can be paused at any point by clicking the Pause button Cc Always pause the run before opening the sample drawer Pausing the run will enable the Stop and Edit buttons Clicking the Stop button LJ will cancel the remainder of the protocol and the software will transition to the post run screen Clicking the Edit button ty will transition the application to the protocol editing screen and will automatically de select all of the blocks that ran to completion before the Edit button was pressed This allows the parameters of a run to be edited before the run finishes When the Run button is clicked from the Edit screen the run will resume from the beginning of the block that was in progress when the run was paused The event log records events that occur during a run To add a note to the event log use the Add comment button MN below the Event Log After the run The post run screen Figure 19 displays the most recently completed run s event log and any images that were acquired The Home button 18 at the top of the screen will return

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