Thermo Scientific Invitrogen E-Gel A33811 Скачать руководство пользователя страница 46

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E-Gel

™

Power Snap Electrophoresis System User Guide

Appendix E

Running RNA Samples on E-Gel

™

EX Agarose Gels

E-Gel

™

EX Agarose Gels can be used to run RNA samples. RNA can be run under denaturing or non-denaturing

conditions. Use non-denaturing conditions only when checking for RNA quality, where accurately determining size
is not critical.

Important

: Using other denaturing agents like Glyoxal, Formaldehyde, or Urea results in very poor separation and

band morphology on E-Gel

™

EX.

It is not recommended to run samples that were loaded with RNA loading buffer on the same gel as samples that are

loaded with water.

Non-

denaturing
conditions

•

Mix RNA sample with RNase-free water such that the final volume is 20 μL.

•

Do not heat. Load the entire sample onto the E-Gel

™

EX.

•

Run RNA using the E-Gel

™

EX 1−2% program for 10 minutes.

Denaturing

agents

The only denaturing agent that is compatible with the E-Gel

™

EX system is Formamide, 50–95%.

Lower concentrations are also acceptable.

Denaturing
conditions

There are two methods for denaturing your RNA sample to run on an E-Gel

™

EX Agarose Gel.

Method 1

Mix RNA (250 ng–2 μg) sample with formamide (to 50–95%) such that the final volume is 20 μL.

Heat samples at 65°C for 5 minutes to denature RNA.

Place samples on ice immediately after heating.

Load entire sample onto E-Gel

™

EX.

Run RNA using the E-Gel

™

EX 1−2% program for 10 minutes.

Method 2

Mix RNA (250 ng–2 μg) sample with RNAse-free water or loading buffer such that the final

volume is 20 μL.

Heat samples at 65°C for 5 minutes to denature RNA

Содержание Invitrogen E-Gel A33811

Страница 1: ...ap Camera For use with E Gel E Gel EX E Gel Go CloneWell and SizeSelect agarose gels Catalog Numbers G8100 G8200 G8300 G8141ST G8142ST G8151ST G8152ST G8168ST G8162ST G8341ST G8342ST G8351ST G8352ST and A33811 Publication Number MAN0017050 Revision B 0 For Research Use Only Not for use in diagnostic procedures ...

Страница 2: ...ITH OR ARISING FROM THIS DOCUMENT INCLUDING YOUR USE OF IT Revisionhistory Revisionhistory of Pub No MAN0017050 Revision Date Description B 0 08 April 2020 Corrections throughout manual A 0 7 September 2017 First version Important Licensing Information This product may be covered by one or more Limited Use Label Licenses By use of this product you accept the terms and conditions of all applicable ...

Страница 3: ...Camera 12 User graphical interface overview 13 Using the E Gel Power Snap Electrophoresis Device 14 Required materials 14 Prepare samples 14 Dilute samples containing high salt 15 DNA ladder preparation guidelines 15 Prepare gel 16 Prepare E Gel Go Agarose Gel 16 Sample loading guidelines 17 Load samples 17 Run the gel 18 Check status 19 View gel 19 View gel with filter lid open 19 Modify a run 19...

Страница 4: ... Prepare samples 25 Prepare gel 25 Load samples 26 Run the gel 26 Check status 26 Prepare wells 26 CollectDNA fragment 27 Guidelines for estimating run time 27 Troubleshooting 28 E Gel SizeSelect II gels 29 Advantages 29 General guidelines 29 Prepare samples 29 Prepare gel 29 Load samples 30 Run the gel 30 Check status 30 Prepare wells 30 CollectDNA fragment 31 Guidelines for estimating run time 3...

Страница 5: ...S cassettes with a Gel Knife 42 Cleaning and storage 43 E Gel Opener 43 Open the E Gel cassette with an E Gel Opener 44 Cleaning and storage 44 E Gel agarose gel disposal guidelines 44 Appendix D 45 Choosing the right DNA ladder 45 Appendix E 46 Running RNA Sampleson E Gel EXAgarose Gels 46 Non denaturing conditions 46 Denaturing agents 46 Denaturing conditions 46 Appendix F 47 E Gel Power SnapBlu...

Страница 6: ...s 53 Appendix H 54 Safety 54 Before starting 54 Installing the instrument 55 Electromagnetic compatibility EMC standards 55 Class A notice 55 Electrical safety 56 Service operation requirements 56 LED Light Emitting Diode 56 Explanation of symbols and warnings 57 Appendix I 58 Customer and technical support 58 Limited product warranty 58 ...

Страница 7: ...ntact Technical Support or download the appropriate QRC from thermofisher com Safety Some commercially available E Gel agarose gels contain ethidium bromide a known mutagen The concentration of ethidium bromide in each gel ranges from 0 1 to 0 3 μg mL All E Gel agarose gels contain 0 055 Proclin added as a preservative Eachgel is provided in a sealed package to protect users from exposure As a pre...

Страница 8: ...tment features The system is optimized for use with E Gel EX E Gel SYBR Safe E Gel Go E Gel CloneWell II and E Gel SizeSelect II gels as well as the E Gel EX Double Comb and E Gel Double Comb with SYBR Safe Features Fast DNA separation in as little as 5 minutes for with E Gel EX Agarose Gels Real time sample view for instant analysis and run control Quick gel image documentation with E Gel Power S...

Страница 9: ...y for any damage incurred during transit File any damage claims with the carrier The warranty does not cover in transit damage Storage E Gel Power Snap Electrophoresis Device Store the devices at room temperature Do not store or use the electrophoresis bases at 4 C E Gel agarose gels Store E Gel pre cast gels ONLY at room temperature Do not allow the temperature to drop below 4 C or rise above 40 ...

Страница 10: ...arts Front view Camera control touch screen USB port for image export firmware upgrade Electrophoresis unit control touch screen Open button for filter lid Lid with amber filter Docking connector cover E Gel Power Snap Camera docked E Gel Power Snap Electrophoresis Device ...

Страница 11: ... upgrade DC input Power switch Docking connector cover Adaptor DC output cable Connector to DC input of electrophoresis unit AC power cord inlet Lid with amber filter open Docking connector cover Electrodes Open button for filter lid Electrophoresis unit control touch screen Blue light transilluminator E Gel cassette compartment ...

Страница 12: ... Power Snap Electrophoresis System User Guide Parts of the E Gel Power Snap Camera Docking connector cover open Docking connector Camera connector Battery compartment USB port for image export firmware upgrade ...

Страница 13: ... Initiate gel run workflow Status dial Switch on off blue light transilluminator Settings screen to access About instrument Screen brightness Software update Service mode Pause Resume gel run Run last protocol select gel protocol E Gel Power Snap Camera controls Status dial to view gel and access Capture gel image Edit adjust capture settings Export image Gallery screen to access Actions screen to...

Страница 14: ... E Gel Power Snap Camera Cat No G8300 E Gel Imager or other third party imager USB storage device not included Prepare samples Sample preparation is critical for separation quality Follow these guidelines for best result Prepare DNA sample in deionized water or 1X E Gel Sample Loading Buffer Use the indicated amount of DNA per well for single or multiple bands If you are unsure how much to use tes...

Страница 15: ...of ladder per well Overloading the ladder will result in distorted or incomplete band separation E Gel DNA Ladder E Gel EX E Gel EX Double Comb E Gel with SYBR Safe E Gel EX Double Comb with SYBR Safe E Gel CloneWell II E Gel SizeSelect II E Gel Go E Gel NGS E Gel Ultra Low Range DNA Ladder 4 μL 100 ng 20 μL 500 ng E Gel 50 bp DNA Ladder 2 μL 50 ng 2 μL 50 ng 20 μL 500 ng 20 μL 500 ng 2 μL 50 ng 1...

Страница 16: ...tte to secure the cassette 5 Load gels within 15 minutes after opening the package Prepare E Gel Go Agarose Gel 1 Remove E Gel Go Agarose Gel from package 2 Gently remove comb from the cassette 3 Place the cassette into the E Gel Go Adaptor 4 Load the adaptor containing the gel into the cassette compartment starting from the right edge 5 Press down on the left side of the cassette to secure the ca...

Страница 17: ...amples by adding E Gel 1X Sample Loading Buffer or deionized water to the required amount of DNA to bring the total required sample volume Avoid introducing bubbles while loading Bubbles can cause band distortion Gel type Total loading volume E Gel EX 20 μL E Gel with SYBR Safe E Gel EX Double Comb E Gel Double Comb with SYBR Safe E Gel Go 10 μL E Gel CloneWell II 25 μL E Gel SizeSelect II 25 μL E...

Страница 18: ...l 4 E Gel EX 4 15 min 20 min E Gel EX Double Comb Agarose Gels 1 and 2 E Gel EX 1 2 5 min 8 min E Gel Agarose Gel with SYBR Safe 1 2 E Gel 0 8 2 26 min 40 min E Gel Agarose Gel with SYBR Safe 4 E Gel 4 30 min 40 min E Gel Double Comb SYBR Safe Agarose Gels 1 and 2 E Gel Double Comb 13 min 18 min E Gel CloneWell II Agarose Gel 0 8 CloneWell 0 8 12 min 40 min E Gel SizeSelect II Agarose Gel 2 SizeSe...

Страница 19: ...Gel SYBR Safe E Gel Double Comb with EX and SYBR Safe dyes E Gel CloneWell II E Gel SizeSelect II and E Gel Go agarose gels For optimal viewing dim the ambient lighting in the room or use the E Gel Power Snap Camera for visualization see page 23 Viewgel 1 Press Back light to activate the blue light transilluminator Note The transilluminator turns off automatically after 1 minute 2 Monitor the samp...

Страница 20: ...ion field to open a keyboard to enter a number 5 Select Resume to restart the run Note Do not run the same gel multiple times or extend the gel protocol beyond the maximum allowed duration Running the gel past the allowed duration will damage the gel and result in poor sample separation Change to another protocol 1 Press Pause run to temporarily stop the run 2 Press the status dial 3 Press Cancel ...

Страница 21: ...amera is started requires thedateandtimetobe set 1 Select Settings 2 Select Instrument settings 3 Select Date Time 4 Choose the date and time format then select Done 5 Set the current date and time then select Done Modify camera settings Access E Gel Power Snap Camera settings from the home screen by pressing Settings Select Instrument setting to adjust screen brightness default image size type an...

Страница 22: ...or 3 Lower the E Gel Power Snap Camera on top of the electrophoresis device and gently snap the camera in place 4 Once connected the E Gel Power Snap Camera displays a brief welcome splash screen which changes to the home screen when it is ready to use Remove the camera 1 Carefully hold the sides of the camera hood and insert your fingers toward the rear of the handhold 2 Lift the camera straight ...

Страница 23: ...Capture to access the capture screen and save image s to the camera 2 Adjust capture settings if necessary Adjust capture settings Settings for the E Gel Power Snap Camera other than exposure can be adjusted during the capture session 1 Press Edit from the capture screen 2 Select the desired image setting from the drop down menu 3 Use or move the slider to adjust the selected setting 4 Press Done ...

Страница 24: ...capture screen Images previously stored on internal memory are accessed from the image gallery Export from capture screen 1 Insert a USB storage device into the USB port at the front of the E Gel Power Snap Camera 2 Press Export from the capture screen 3 Review the images in the active session gallery and select files for export 4 Optional Select Edit info to change the file name file type Jpeg TI...

Страница 25: ...filter lid opened For guidance on disposal of used gels see SYBR Safe DNA Gel Stain page 48 Prepare samples Prepare up to 25 μL of sample in 1X Sample Loading Buffer e g use 2 5 μL of 10X Sample Loading Buffer with 22 5 μL total sample 10X Sample Loading Buffer is provided with E Gel Clonewell II Agarose Gels Use the indicated amount of DNA per well for single or multiple bands Divide samples with...

Страница 26: ...e reference line 4 Run the gel protocol by pressing Start run The run stops automatically after the programmed time has elapsed Check status 1 Check the gel status by activating the Back light Monitor the gel during the run to avoid the target fragment missing the recovery well 2 Pause the gel when the band of interest reaches the reference line RF near the row of recovery wells Important Put on o...

Страница 27: ... the band of interest passes the recovery well see page 20 Guidelines for estimating run time Refer to the E Gel 1 Kb Plus Express DNA Ladder migration pattern table to estimate target DNA run time to the reference line The run times indicated in the table are estimates Monitor your gel in real time during the run to ensure the sample does not pass the recovery well Identically sized bands in diff...

Страница 28: ...epared sample per well Recovery wells were not filled with water prior to elution Once target fragment reaches reference line pause the run and fill all recover wells with deionized water DNA band passed the recovery gel Carefully observe the band migration into the recovery well Minimize ambient light or perform the workflow in dark room DNA band amount is too high Collect DNA from the well in tw...

Страница 29: ...amples Prepare up to 25 μL of sample in 1X Sample Loading Buffer e g use 2 5 μL of 10X Sample Loading Buffer with 22 5 μL total sample 10X Sample Loading Buffer is provided with E Gel SizeSelect II Agarose Gels Use the indicated amount of DNA per well for single or multiple bands Do not exceed 1 μg for sheared DNA Divide samples with higher amounts of DNA across multiple wells Use up to 25 μL tota...

Страница 30: ...ng Start run The run stops automatically after the programmed time has elapsed Check status 1 Check the gel status by activating the Back light Monitor the gel during the run to avoid the target fragment missing the recovery well 2 Pause the gel when the reference band of the DNA ladder reaches the reference line RF near the row of recovery wells Important Put on orange Safe Imager viewing glasses...

Страница 31: ...Ladder is also used as a size reference marker Refer to the NGS library size selection reference to estimate run time from the reference line to the collection well The run times indicated in the table are estimates Monitor your gel in real time during the run to ensure the sample does not pass the recovery well Identically sized bands in different wells may migrate differently DNA fragment size a...

Страница 32: ...ase read 390 bp 13 16 min 500 ng 400 bpband is at the middle of the exposed agarose area 50 100 ng 500 bpband is at the top of the exposed agarose area 200 base read 330 bp 12 14 min 500 ng 350 bpband is at the top of the exposed agarose area 50 100 ng 350 bpband has just completely enteredthe topedge of the collection well 100 base read 200 bp 11 12 5 min 500 ng 200 bpband is in the middle of the...

Страница 33: ...assessed using the Qubit fluorometer Cat no Q32868 or by gel electrophoresis qPCR is recommended for accurate quantitation of next generation sequencing libraries recovered from E Gel SizeSelect II gels Recovered samples are not compatible with 280 nm measurements without first performing buffer exchange ...

Страница 34: ...uffer Low yield Incorrect loading volume chosen Load up to 25 μL of prepared sample per well Recovery wells were not filled with water prior to elution Once target fragment reaches reference line pause the run and fill all recover wells with deionized water Target DNA passed the recovery gel Carefully observe the DNA migration into the recovery well Minimize ambient light or perform the workflow i...

Страница 35: ...el type Total sample volume is too low Load recommended sample volume based gel type Keep all sample volumes uniform Load deionized water in all empty wells Physical gel damage Avoid touching the gel well with the pipette when loading the sample Band distortion caused by air bubbles Avoid introducing bubbles while loading the samples Gel was not electrophoresed immediately after sample loading Run...

Страница 36: ...boptimal or no image when used with E Gel Power Snap Camera Incorrect camera adjustments Refer to E Gel Power Snap Camera use guide High background suboptimal or no image when used with E Gel Imager No filters or wrong filter set Refer to E Gel Imager Technical Guide or instrument manufacturer for optimal filter set Photographic settings not optimal Determine optimal settings empirically by adjust...

Страница 37: ... 1 Download the latest firmware file from thermofisher com to your PC 2 Unzip and transfer the firmware upgrade files to a USB storage device 3 Insert the USB storage device into a USB port on the instrument a Use the port located at the back of the E Gel Power Snap Electrophoresis Device A to upgrade the electrophoresis unit b Use the port located at the front of the E Gel Power Snap Camera B to ...

Страница 38: ...partment Instrument Specifications Instrument dimensions and specifications Specification E Gel Power Snap Electrophoresis Device Dimensions 242 mm 130 mm 70 mm Weight 1 kg Touchscreen LCD display 77 4 mm 43 86 mm Viewing surface dimensions 90 mm 110 mm Amber filter dimensions 86 mm 105 mm LED light Blue LED CWL 465 nm FWHM 20 nm LED life 50 000 hours LED specification Array of 12 high power LEDs ...

Страница 39: ...etic radiation may interfere with the proper operation of the device Altitude Between sea level and 2000 m 6500 ft above sea level Operating conditions Humidity 15 80 relative humidity noncondensing Temperature 15 to 30 C 59 to 86 F Note For optimal performance avoid rapid or extreme fluctuations in room temperature Storage and transport conditions Humidity 20 80 relative humidity noncondensing Te...

Страница 40: ...stem a pH balancing system and DNA stainpackaged inside a transparent plastic cassette Each gel cassette contains two ion exchange matrices IEMs that are in contact with the gel and electrodes The IEMs supply a continuous flow of ions throughout the gel resulting in a sustained electric field required for running the gel Comb Sample wells Upper ion exchange matrix Cathode Agarose gel Lower ion exc...

Страница 41: ... range 1 100 bp 5kb 2 50 bp 2kb 4 10 bp 500bp 1 100 bp 5kb 2 50 bp 2kb 1 100 bp 5kb 2 50 bp 2kb 4 10bp 500bp 1 100 bp 5kb 2 50 bp 2kb 1 100 bp 4kb 2 50 bp 2kb Run time 1 2 10 20min 4 15 20min 1 2 5 8min 1 2 26 40min 4 30 40min 1 2 13 15min 15 30min Access to sample Yes openable No Gels for preparative gel electrophoresis in Cloning and NGS applications E Gel CloneWell II E Gel Size Select II E Gel...

Страница 42: ...ant Before opening the E Gel cassette put on safety goggles and gloves Gel Knife The Gel Knife Cat no EI9010 is used to open the cassette for E Gel EX and E Gel NGS agarose gels Open E Gel EX and NGS cassettes with a Gel Knife 1 Place the cassette on a flat surface with the wells facing upward 2 Insert the sharp edge of the gel knife into the groove around the edge of the cassette edge then lever ...

Страница 43: ...cassette so the gel can be removed for staining excision of DNA fragments or blotting The E Gel Opener consists of an anodized aluminum platform housing two recessed steel blades one which is stationary and one which is movable Before using the E Gel Opener for the first time we recommend that you practice opening a few used E Gels that will not be used further for preparative purposes to familiar...

Страница 44: ...ll damage the E Gel agarose gel 6 Unscrew the knob and remove the cassette You may have to carefully work the cassette from the housing because the cassette fits snugly in the recessed groove 7 Turn the cassette 90 and re insert the cassette into the E Gel Opener to open the two remaining sides 8 Repeat steps 4 5 to break the two remaining seals 9 Unscrew the knob and carefully remove the E Gel ca...

Страница 45: ...ative DNA Ladder E Gel Ultra Low Range DNA Ladder Millenium RNA Marker Century Plus RNA Ladder E Gel Sizing DNA Ladder Gel Type Agaros e Cat No 10488090 Cat No 10488091 Cat No 10488099 Cat No 12352019 Cat No 12373031 Cat No 10488096 Cat No AM7150 Cat No AM7145 Cat No 10488100 E Gel Agarose Gels w SYBR Safe DNA Stain 1 2 4 E Gel Double Comb Agarose Gels w SYBR Safe DNA Gel Stain 1 2 E Gel EX Gels 1...

Страница 46: ...ee water such that the final volume is 20 μL Do not heat Load the entire sample onto the E Gel EX Run RNA using the E Gel EX 1 2 program for 10 minutes Denaturing agents The only denaturing agent that is compatible with the E Gel EX system is Formamide 50 95 Lower concentrations are also acceptable Denaturing conditions There are two methods for denaturing your RNA sample to run on an E Gel EX Aga...

Страница 47: ... Snap Electrophoresis Device transilluminator is designed for viewing E Gel with SYBR Safe gels E Gel EX gels E Gel CloneWell II gels E Gel SizeSelect II and E Gel Go gels The use of blue light transillumination is advantageous over the UV as it does not require UV protective equipment during use In preparative gel electrophoresis blue light transillumination results in dramatically increased clon...

Страница 48: ...ella strains and only with activation by a mammalianS9 fraction Produces no signs of mortality or toxicity at a limit dose of 5000 mg kg from a single oral administration View studies documenting the safety of SYBR Safe in the SYBR Safe White Paper document available from thermofisher com content dam LifeTech global life sciences pdfs 494 pdf Cloning benefits By using the blue light transilluminat...

Страница 49: ...thidium bromide containing gels Contact your safety office or local municipality for appropriate disposal in your community Spectrum When bound to nucleic acids the proprietary nucleic acid stain in E Gel EX E Gel SizeSelect and E Gel Go agarose gels has fluorescence excitation maxima at 490 nm and an emission maximum at 522 nm see figure below Normalized fluorescence excitation and emission spect...

Страница 50: ...luded Equipment Cat No E Gel Power Snap Electrophoresis Device E Gel Power Snap Camera Power Cord w Adaptor Safe Imager Viewing Glasses Cat No S37103 Gel Knife G8141ST 1 each 1 each 1 each 1 each G8142ST G8151ST G8152ST G8168ST G8162ST G8131ST G8132ST G8171ST G8172ST G8341ST 1 each 1 each G8342ST G8351ST G8352ST G8331ST G8332ST G8371ST G8372ST ...

Страница 51: ...afe Gel 2 18 100 apps G8168ST E Gel CloneWell II Gel 0 8 10 100 apps G8162ST E Gel SizeSelect II Gel 2 10 100 apps G8131ST E Gel EX Double Comb 1 10 100 apps 100 apps G8132ST E Gel EX Double Comb 2 10 100 apps 100 apps G8171ST E Gel DC with SYBR Safe 1 10 100 apps 100 apps G8172ST E Gel DC with SYBR Safe 2 10 100 apps 100 apps G8341ST E Gel EX Gel 1 10 100 apps G8342ST E Gel EX Gel 2 10 100 apps G...

Страница 52: ...rose Gels 1 10 gels G441001 20 gels G442001 2 10 gels G441002 20 gels G442002 E Gel NGS Agarose Gels 0 8 10 gels A25798 E Gel CloneWell II Agarose Gels 0 8 10 gels G661818 E Gel SizeSelect II Agarose Gels 2 10 gels G661012 Accessoryproducts E Gel DNA Ladders Quantity Applications Catalog No E Gel 1 Kb Plus DNA Ladder 25 ng μL 2 x 1 mL 100 apps 10488090 E Gel 1 Kb Plus Express Ladder 40 ng μL 2 x 1...

Страница 53: ...E Gel Power Snap Electrophoresis System User Guide 53 Accessoryitems Product Quantity Catalog No Safe Imager Viewing Glasses 1 each S37103 Gel Knife 1 each EI9010 E Gel Opener 1 each G530001 ...

Страница 54: ...red skills be allowed to use the product Keep the basic safety instructions and the product documentation in a safe place and pass them on to the subsequent users Applicable local or national safety regulations and rules for the prevention of accidents must be observed in all work performed Operation of the E Gel Power Snap Electrophoresis System is subject to the following conditions Indoor use A...

Страница 55: ...k fire and or serious personal injury or death The product is for indoor use only Use only with suitably rated mains supply cord having 3 conductors min 16 AWG or 1 5 mm2 min 300V Harmonized Type for Europe and UL Listed CSA Certified for North America with molded plug rated min 10A A tolerance of 10 shall apply to the nominal input voltage and 3 Hz to the nominal frequency overvoltage category 2 ...

Страница 56: ...cket Otherwise sparks that result in fire and or injuries may occur Do not overload any sockets extension cords or connector strips doing so can cause fire or electric shocks Ensure that the connections with information technology equipment e g PCs or other industrial computers comply with the IEC60950 1 EN60950 1 or IEC61010 1 EN 61010 1 standards that apply in each case Unless expressly permitte...

Страница 57: ...am Turn off the lamp before opening Use eye protection during servicing Potential biohazard Protective conductor terminal main ground On Off WEEE Do not dispose of this product in unsorted municipal waste CAUTION To minimize negative environmental impact from disposal of electronic waste do not dispose of electronic waste in unsorted municipal waste Follow local municipal waste ordinances for prop...

Страница 58: ...ding User guides manuals and protocols Certificates of Analysis Safety Data Sheets SDSs also known as MSDSs Note For SDSs for reagents and chemicals from other manufacturers contact the manufacturer Limited product warranty Life Technologies Corporation and or its affiliate s warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologie...

Страница 59: ...For support visit thermofisher com techresources or email techsupport lifetech com thermofisher com lifescience 08 April 2020 ...

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