Operation of the Covaris E
Contents
1. Non Controlled Version Note Controlled Versions of this document are subjected to change without notice LOAD SAMPLE PLATE TR Figure 10 Sample Plate Positioning 2 4 4 Close the door and click the Check Alignment button see Fig 2 Make sure that the stage with samples moves to the front left of the water bath and lowers down into he water which is the position of the plate during processing Al position well should be located above the transducer 2 4 4 1 1f the stage moves to the back right it means that running parameters have not been set yet Select the shearing parameters see section 2 2 and then click Check Alignment button again The stage should move to the front left side of the water bath 2 4 5 With the plate holder situated in the front left position of the water bath the processing position check the water level For microTUBEs the water level must be at the very TOP of black plate holder See Figure 11 ee ee Operation of the Covaris E Series ClEnCES er i Saved as LIBPR 0041 Supersedes Version 3 Page 12 of 15 Non Controlled Version Note Controlled Versions of this document are subjected to change without notice Figure 11 Red arrow indicates the water level for shearing samples using microTUBEs 2 4 6 For microTUBE plates the plate sites higher on the stage than the microTUBEs so the water level must higher near the top edge of the microTUBE plate Use a dummy2. 4 9 The water conditioning unit located to the right of the instrument requires bi weekly change of water Leave the water conditioning system ON
3. O rn V eee ee Operation of the Covaris E Series K eee a 7 VCiences Centre Saved as LIBPR 0041 Supersedes Version 3 E Version 4 Page 1 of 15 Non Controlled Version Note Controlled Versions of this document are subjected to change without notice Operation of the Covaris E series I Purpose To provide instructions for operation and maintenance of Covaris E series To specify conditions for shearing DNA to the appropriate size range for library construction II Scope All procedures are applicable to the BCGSC TechD and Library Core groups III Policy All production procedures shall be documented and controlled by approved systems IV Responsibility It is the responsibility of all personnel performing this procedure to follow the current protocol It is the responsibility of the Production Coordinator to ensure personnel are trained in all aspects of this protocol It is the responsibility of Quality Assurance Management to audit this procedure for compliance and maintain control of this procedure V References Reference Title Reference Number Covaris E series User Manual IM 0161 VI Related Documents N A VIL Safety All Laboratory Safety procedures will be complied with during this procedure The required personal protective equipment includes a laboratory coat and gloves See the material safety data sheet MSDS for additional information VIII Materials and Equipment Supplier Nu
4. Lower the plate holder into the water chamber by clicking the Check Alignment icon on the screen See Figure 4 Load Sample Check Alignment Bath Temperature CANAD A S MICHAEL CIEWCES SMITH LEWLVE www becgsc ca Operation of the Covaris E Series Saved as LIBPR 0041 Supersedes Version 3 Page 5 of 15 Non Controlled Version Note Controlled Versions of this document are subjected to change without notice KOCK RK Ke OCH E A A D 0g o 2 6 0 0 6 0 6 6 RG ae Oh OR 224822822 5 60 000 000 i An N i y io Ve _ Figure 4 Main Panel on Covaris E series 4 7 4 A f 4 V AA Y ALN 1 N 1 10 With the stage in a check alignment position front left of the water chamber with A A dd Y f position A1 of the plate above the transducer confirm tank water level using the A i X l sticker on the front glass l Wm s 4 a Ve 4 A Wr A 4 1 10 1 For microTUBEs adjust water 4 A l y Es adjust level to level 10 RUN or 15 FILL 1 10 2 For microTUBE plates adjust water level to 6 RUN or 12 FILL See Figure 5 wi 1s 15 4 n S D A 0 0 microTUBE plate RUN 6 WATER D N TA N N N ae s XY Vv Figure 5 Water level indicator with stage at shearing check
5. discuss with supervisor regarding sonication conditions 2 3 2 For tube based library construction 40uL volume 2 3 2 1 For Genome shotgun libraries 400bp 20 duty cycle Intensity 5 200 bursts per minute time 45sec 2 3 2 2 For Exome Capture libraries 200 250bp 20 duty cycle Intensity 5 200 bursts per minute time 75sec 2 3 2 3 For Transcriptome libraries 200 300bp 20 duty cycle Intensity 5 200 bursts per minute time 75sec Note Some samples require different shearing conditions than the ones listed above consult with supervisor if different conditions are to be used 2 3 3 Click Return This maintains the template file as set by your APC It is not necessary to save your configuration in order to run it If you wish to save your configuration click Save and gave the file a new name so that the production template remains unchanged 2 4 Sample Loading and shearing 2 4 1 To load samples click on the Load Sample icon see Fig 2 2 4 2 When the stage stops moving the Door button will light up in green To open the door press on the green lighted Door button and while depressed pull the door handle firmly 2 4 3 Place the plate containing covaris tubes in the plate holder with sample A1 of the plate on the far right See Figure 10 V T gt M E a Operation of the Covaris E Series A Centre Saved as LIBPR 0041 Supersedes Version 3 _ Page 11 of 15
6. applications will help prevent corrosion and ensures optimal system performance Close the door See Figure 13 ee ee Operation of the Covaris E Series a N aM hiha Saved as LIBPR 0041 Supersedes Version 3 a Page 15 of 15 Non Controlled Version Note Controlled Versions of this document are subjected to change without notice gt SonoLAG File Ed Process Hep Covaris adaptive Focused scowsiics Pro essRunning Stop Button SonoLAB E v4 3 3 Motors Moving Safety Interrupt Sample Preparation System Deel ee Change Bath Active Process 1con Current Treatment Acoustic Power Duty Cycle Intensity Cydes Burst 20 Start we Zt z ig Ly ie ai le Current Sample z f i hFa _ Current Position Ci 2 Offset 0 0 mm st ton i a Estimated Time to Completion Progress 9 5 Eyit 0 10 20 30 40 50 60 70 BO 320 100 2003 Covaris Inc Figure 13 Shutdown of Covaris E series 4 5 Press Exit icon in the lower right corner of the SonoLab application A message will ask you to confirm the status of the shutdown Make sure that all of the listed steps in that window are completed 4 6 Close the SonoLab software by selecting Exit on the main panel 4 7 Turn off the computer from the START menu and close the case 4 8 Power down the Covaris E series by turning power switch button located on the right of the instrument to O position
7. alignment position a lt 7 N 1 11 If necessary adjust the water bath level with deionized or distilled water If water level is changed the water bath needs to be degassed for an additional 20 minutes ee ee Operation of the Covaris E Series ClEnCES er i Saved as LIBPR 0041 Supersedes Version 3 Page 6 of 15 Non Controlled Version Note Controlled Versions of this document are subjected to change without notice 1 12 The Covaris E series system requires that the water bath be degassed to maintain repeatable acoustic energy coupling Press the Degas button and make sure that the degas light is on see Fig4 below Check that there are bubbles coming from the end of the degassing tube 1 13 At room temperature the water bath should be degassed for at least 30 min prior to running the shearing process Degas light should appear yellow Door button must be depressed to open the door to load sample plate Figure 6 Degas button location It should be yellow when active Wait until the bath temperature indicator on the screen shows temperature below 12 C 1 14 The system can be switched on and water bath degassed first thing in the morning and then kept on during the entire working day or entire working week The system should be switched off over the weekend 2 Running of samples 2 1 Sample Preparation 2 1 1 For manual tube based library construction use P200 pipette to load 60UWL of sample res
8. between these will be highlighted Press DELETE Confirm the wells remaining match your samples See Figure 9 Note Forcreatinga new shearing process click Configure icon instead CANAD A S MICHAEL CIEWCES www becgsc ca SMITH LEWLVE Operation of the Covaris E Series Saved as LIBPR 0041 Supersedes Version 3 Page 9 of 15 Non Controlled Version Note Controlled Versions of this document are subjected to change without notice Process Confieuration Plate based JUsec_Production_11i040 Ble Edt Help Covaris well F Duty Cycle Intensity Cycles burth Treatment Time I Radus WV Rate Dwell Hiz a J 1000 fis0 0 Foo mm o0 rpm 0 0 5 10 am or l ae son Well conditions mer m ta T oe ap K war A n r d ih f AC HE i T om O ices 4 li AA AA A oie P p gt at a Bg JE fae a rT ai 5 a a a L mis ka L i 1 T a 1 g i L A kuge TSR EA KA E ia gi a s j i tae 2 i i i Eh Rhi A oak TE F HAA a i k k PaPa Ta P 5 LAF A r ak f ho Re et a SiB EBBS BB ElS 8 888 8E 888818 E8888 Figure 9 Covaris E series Process Configuration Screen 2 2 4 Every production process will show at least one well set up with the appropriate shearing parameters for the desired size f
9. mber Model or Catalogue E110 CANAD A S MICHAEL SMITH Operation of the Covaris E Series a LE ME Saved as LIBPR O041 Supersedes Version 3 _ Page 2 of 15 Non Controlled Version Note Controlled Versions of this document are subjected to change without notice Deionized water In house N A MicroTube 6x 16mm with crimp cap Covaris 520052 Eppendorf P200 Pipette Eppendorf P200 IX Procedure 1 Equipment preparation 1 1 Unplug any other equipment from the outlet where the Covaris E series will be operating Plug in the Covaris E series 1 2 Water conditioner located to the right of the instrument should already be on 1 3 Start the E210 by setting the power switch located on the right of the instrument to the 1 position Do not start the chiller bath See Figure 1 Figure 1 Covaris E series 1 4 Turn on the computer attached to the Covaris E series Click on the SonoLab icon to open the software 1 5 Confirm the Intensifier is installed If absent it must be installed before you continue Refer to step 1 5 If it is present skip to Step 1 6 1 5 1 Install intensifier in E210 ee Operation of the Covaris E Series A em Me Saved as LIBPR 0041 Supersedes Version 3 Page 3 of 15 Non Controlled Version Note Controlled Versions of this document are subjected to change without notice 1 5 1 1 Wait for the homing sequence to complete Select the CHANGE BATH button The tran
10. mly 2 4 13 Remove the plate from the holder Close the door 2 4 14 Covaris can be left turned on during the working day If you need to shut it down proceed to step 5 0 3 Removal of samples from covaris tubes 3 1 For plate based library construction refer to LIBPR 0045 DNA Transfer to Covaris Tube Rack and Sheared DNA Transfer to 96 well Plate Using the Biomek FX SOP to transfer samples out of covaris tubes into a 96 well AB1000 plate 3 2 For tube based manual library construction the samples can be withdrawn from the tubes using P200 pipette 3 2 1 Spin down the covaris tube in a minifuge 3 2 2 To withdraw the sample angle the pipette tip while perforating the septum Sample withdrawal can be difficult Repeat pipetting out until the entire sample is withdrawn Ifa vacuum persists inserta new P2 tip alongside the P200 tip so air can enter the micro TUBE Do not use the same P2 or P200 tip on more than one tube or the samples will be cross contaminated 4 Shutdown and Maintenance 4 1 To shutdown the Covaris E series turn off the chiller located under the bench 4 2 Water conditioning system should be left turned ON 4 3 Press the Change Bath icon to raise the transducer out of the water bath 4 4 Open the door and with extreme care remove most of the water from the transducer bowl with a pipette Then using soft lint free cloth paper sponge remove any residual water Keeping the transducer dry between
11. plate provided beside the Covaris E210 to test water level See Figure 12 microTUBE plate sealed with Clear Film Plate is partly submerged Water level just below top edge of plate oes A bene Ye aoa Operation of the Covaris E Series sd ce eee VCiences Centre Saved as LIBPR 0041 Supersedes Version 3 Ea Version 4 Page 13 of 15 Non Controlled Version Note Controlled Versions of this document are subjected to change without notice Figure 12 Red arrow indicates the water level for shearing samples using microTUBE plate 2 4 7 If water level needs adjustment then without removing the plate open the door press on the green lighted Door button and while depressed pull the door handle firmly and add more distilled or de ionized water or if necessary remove the excess The sample plate should NOT be completely submerged IMPORTANT NOTE If any water addition is done at this point water bath needs to be degassed again for at least 30min During this time your samples can be kept submerged in the water bath If any water removal is done no additional degassing is necessary 2 4 8 On the SonoLab software screen click on the LOAD icon to swap in your sample plate and press CHECK ALIGNMENT icon If the water level does not need to be adjusted set your timer for 10 min This ensures that the temperature of samples equilibrates with the temperature of the water bath This step is important becau
12. raction Without changing of the acoustic parameters copy or delete the existing well conditions to match the number and plate location of your samples For a full plate you should see the same parameters displayed 96 times from well Al to well H12 To remove wells select the top most unused well then hold the SHIFT key and scroll to the last unused well Then press DELETE key to remove them from the table 2 2 5 You can also apply the conditions to your sample wells by clicking on the wells in the plate map shown in Well Plate display on the left See Figure 6 Added wells should appear in the well conditions table 2 3 Sonication conditions are as follows 2 3 1 For plate based library construction 60uL volume ee Operation of the Covaris E Series G E N M E ScTences Centre Saved as LIBPR 0041 Supersedes Version 3 Page 10 of 15 Non Controlled Version Note Controlled Versions of this document are subjected to change without notice 2 3 1 1 For 400bp mean DNA fragment size 20 duty cycle Intensity 5 200 bursts per minute time 30 sec setting used for Illumina genomic samples 2 3 1 2 For 300bp mean DNA fragment size 20 duty cycle Intensity 5 200 bursts per minute time 55 seconds setting used for WTSS samples 2 3 1 3 For 200bp mean DNA fragment size 20 duty cycle Intensity 5 200 burst per minute time 75 seconds 2 3 1 4 For amplicon samples or samples for exon tiling
13. sducer and rack holder will home allowing easy access to the Intensifier See Figure 2 o 6 8 ee SAOOOe Ses 1 Figure 2 SonoLA DAEA Ves ve i button P move raise stage amp transducer 1 5 1 2 Gras AA ot plast ss and betty press the entire assembly into the tiucer well Note the UP label on the transducer Do ot push ome steel cont onthe wires La 3 The rhe isa friction fiti dn the well no hardware is used to hold it in place ate r amp 3 4 AJ N A 7 V ee Operation of the Covaris E Series ClEwCES Cm Me Saved as LIBPR O041 Supersedes Version 3 Page 4 of 15 Non Controlled Version Note Controlled Versions of this document are subjected to change without notice Q Stat _ S a Figure 3 Install Covaris E 210 Intensifier NEVER HOLD BY ITS METAL WIRES sal a co 4 a 1 6 The E series should always have water in the tank circulating through the attached water conditioner Only distilled or deionized water should be used to fill the water bath or adjust the level of the water bath using tap water or any other solution is a misapplication of the equipment and will void the warranty 1 7 Turn on the water chiller located under the bench Make sure the temperature of the chiller is set to 4 C 1 8 Lower the transducer into the water chamber by clicking Load sample icon on the main panel of SonoLab screen 1 9
14. se temperature affects the effectiveness of acoustic waves and this equilibration will help to reduce shearing variability 2 4 9 After 10 min temperature equilibration click the Configure icon and double check the shearing parameters for your samples Once parameters are confirmed click on the Start icon 2 4 9 1 The time to completion is tracked on the screen set your timer to know when to comeback Stay in the room until the second well is being processed to make sure that the shearing is progressing from sample to sample 2 4 10 After completion of all samples there will be a process completion window displayed on the monitor screen If you come back and don t see that window it may mean that the process was never started or it was aborted You will have to perform additional QC of your samples to make sure that all samples were sheared e T E gt M E 7 Operation of the Covaris E Series A mo Centre Saved as LIBPR 0041 Supersedes Version 3 _ Page 14 of 15 Non Controlled Version Note Controlled Versions of this document are subjected to change without notice 2 4 11 In the Process Completion Window click OK and press Load Sample icon on the screen The sample plate will be lifted from the water and brought closer to the door 2 4 12 When the plate holder stops moving the Door button will light up in green Press the green Door button and while depressed pull the door handle fir
15. sed library construction is 5ug ae TA 2 2 Set up of shearing parameters 2 2 1 On the SonoLab software screen click on the Open icon Open Production Folder and select the right shearing program for your size fraction See Figure 7 Figure 7 Covaris SonoLAB GUL Click OPEN button and select the library specific protocol 2 2 2 From the Well Plate menu select tube or plate format See Figure 8 a eee ee V N V S Operation of the Covaris E Series operation of the Covaris E Series Cc ee a e VCiences Centre Saved as LIBPR 0041 Supersedes Version 3 Ea Version 4 Page 8 of 15 Non Controlled Version Note Controlled Versions of this document are subjected to change without notice 2 2 2 1 For microTUBE select 500143 96 position 6mm tube wpl 2 2 2 2 For microTUBE plate select 520078 96 microTUBE plate wpl Covaris Covaris Sequence Sequence o al Pe Ge Vertical Offset User Entered Vertical Offset 7 0 mm 4 0 mm Click Action Append Click Action Append Well Plate nj 500143 96 position 6mm tube Well Plate i520078 96 microTUBE plate wpl Figure 8 Select micro TUBE or microTUBE plate format from Well Plate menu 2 2 3 From the Configure menu select only wells containing sample Select the wells not used by clicking the first well then hold the SHIFT key and scroll to the last unused well and select it All wells in
16. uspended in Qiagen Elution Buffer Tris HCl pH 8 0 Water cannot be used to resuspend a sample for Covaris E series shearing 2 1 1 1 Use one microtube with crimp caps for each sample Tilt the pipette tip on an angle and pierce the septum of the microtube Dispense the sample slowly into the microTUBE Make sure to spin it down to collect sample at the bottom of ee Operation of the Covaris E Series Chew CES aM Me Saved as LIBPR 0041 Supersedes Version 3 Page 7 of 15 Non Controlled Version Note Controlled Versions of this document are subjected to change without notice the tube Place the microTUBEs in the Covaris tube rack with your first sample in the top right position of the rack A1 which has an angled corner Place the remaining tubes in their respective locations Firmly snap the covaris plate cover on top of the tubes ensuring the 4 pins are holding the microtubes in place y 2 1 2 For plate based library construction refer to LIBPR 0045 DNA Transfer to Covaris Tube Rack and Sheared DNA Transfer to 96 well Plate Using the Biomek FX SOP to load 60uL of sample well resuspended in Qiagen EB buffer Tris HC1 pH 8 0 A1 well on the plate should correspond to the top right position A1 of the Covaris tube rack which has an angl d corner Make sure to spin down the plate to collect samples at the bottom of the tube before it is processed 2 1 3 The upper limit of DNA in on sample to shear for both manual and plate ba
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