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DNA Size Selection System

Operations Manual

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Содержание PippinHT

Страница 1: ...DNA Size Selection System Operations Manual...

Страница 2: ...MA 01915 2018 Sage Science Inc All rights reserved Sage Science and PippinHT are trademarks of Sage Science Inc All other brands and name mentioned herein are property of their owners The PippinHT in...

Страница 3: ...4 1 Unpacking the PippinHT 4 1 4 2 Setting up the PippinHT 4 1 4 3 Unpacking Gel Cassette Kits 4 3 4 3 1 Storage Conditions for Cassettes and Reagents 4 4 5 WORKFLOW SUMMARY 5 5 6 SAMPLE PREPARATION...

Страница 4: ...12 2 1 Gel cassette barcodes 12 2 12 2 2 Sample ID entry 12 3 12 2 3 Importing Sample IDs 12 3 12 2 4 Using the Sample ID Template 12 5 12 2 5 Log Files 12 6 12 2 6 Log File Name Format 12 7 12 2 7 A...

Страница 5: ...se Procedure 17 7 18 WARRANTY AND SERVICE 18 1 19 INSTRUMENT SPECIFICATIONS 19 1 20 GEL CASSETTES SPECIFICATIONS 20 1 20 1 3 Agarose 100 bp 250 bp Marker 30G 20 2 20 2 2 Agarose 100 bp 600 bp Marker 2...

Страница 6: ...rrays and a single board computer The system computer is accessed by an external LCD monitor mouse and keyboard Figure 1 1 Software System software allows the user to o Enter parameters that define th...

Страница 7: ...ragments Note that the optical detectors monitor the odd numbered lanes only Accordingly each lane pair must run the same size selection protocol If an external marker strategy is used the marker must...

Страница 8: ...m that lane once the run is complete See section 12 4 and 12 5 1 3 Collection Strategies Collection timing start duration and end is based on the factory calibration settings provided with a cassette...

Страница 9: ...n the resolution of the agarose gel concentration within the cassette The distribution is Gaussian and its range is typically 16 CV 8 of the median fragment size 2 Range Size Distribution Range settin...

Страница 10: ...lecular weight collections using High Pass protocols with 0 75 agarose cassettes These recommendations are outlined in section 2 3 below 2 1 Common Misconceptions Users should be aware of the followin...

Страница 11: ...ration of the time and base pair difference between the detector and branch point 2 1 3 The rate of electrophoresis is faster in the separation channel than in the elution channel This may cause misco...

Страница 12: ...d for HMW DNA High Pass Protocols Recovering high molecular weight DNA from High Pass protocols tend to have lower yields than low molecular weight size selection The following additional steps are hi...

Страница 13: ...ovides additional information regarding the function of the system or applications for which is used Caution Class I invisible Laser Radiation Present Avoid long term viewing of laser Safe Use Guideli...

Страница 14: ...ly o Power cord o Mouse Pad o Operations Manual o USB Memory Stick o Two rinse cassettes for maintenance of electrodes o Two calibration fixtures for setting optical baseline before each run 4 2 Setti...

Страница 15: ...wered down and restarted to set the correct screen resolution 10 Connect the computer keyboard to any USB port located in the back of the Instrument 11 Connect the computer mouse to the PippinHT into...

Страница 16: ...oresis Buffer Package 2 2 x 600 l of loading solution packaged separately for size selection using an external marker 0 75 agarose cassettes uses external markers only 500 l fluorescein labeled marker...

Страница 17: ...ture in the foil bags until ready for use DNA Internal standards loading solution mix Store at 4 C Equilibrate to room temperature before use Return to 4 C immediately after use Electrophoresis buffer...

Страница 18: ...arker 3 2 1 5 cassettes or bring external marker solution to RT 0 75 High Pass cassettes max 11 samples cassette external marker Program a Size Selection Protocol Select USE INTERNAL STANDARDS Program...

Страница 19: ...oteinized prior to loading whenever possible Input DNA size distribution A knowledge of the input size distribution is obviously important to program accurate size selection settings PippinHT cassette...

Страница 20: ...ker as described below Prepare the external marker A For 3 2 and 1 5 agarose gel cassettes 1 Bring DNA standard loading solution mix to room temperature return to 4 C immediately after use 2 For each...

Страница 21: ...rotocols for DNA size selection A protocol is created for a cassette type within the applicable target range and is saved as a named protocol file with a phprot file extension When running a cassette...

Страница 22: ...ds will appear in the size selection parameters Tight collects the narrowest fragment distribution that can be collected by the system The user enters a target base pair value the median for the distr...

Страница 23: ...d whenever the protocol is used If sample information is entered or imported into the Sample ID field in the Main screen then the Sample ID Template names will prefix the Sample ID names LED On The LE...

Страница 24: ...te Previously save protocols may be selected and opened from the protocol pop up window Theses may be edited and saved or resaved with a new file name Any changes to a protocol or the default screen w...

Страница 25: ...ect to load the definition Note Size selection parameters may not be entered until a Cassette Type Definition has been selected Note A run time value is displayed This value is set by the cassette def...

Страница 26: ...t a wider range to be collected using starting and ending base pair values Note Range mode is used for programming High Pass protocols Time allows users to program extractions using the starting and e...

Страница 27: ...Press COPY LANE 1 TO CASSETTE to copy the size selection parameters to the remaining lane pairs on Cassette 1 Note Each lane pair may be programmed separately with different parameters Only lanes 1 2...

Страница 28: ...ng 10 Press COPY CASSETTE 1 TO CASSETTE 2 to copy the size selection parameters including the Sample ID Template information applied to cassette 1 to cassette 2 Note Cassette 2 may be programmed separ...

Страница 29: ...detected lane any odd numbered lane and the remaining lane pairs will use the marker to calibrate the elution timing For 0 75 agarose gel cassettes the marker is provide pre made and ready to load th...

Страница 30: ...d for an external marker may be used for size selection as well provided Start value not the Target value is higher than any other Start value in the remaining lanes Users should be very careful when...

Страница 31: ...ff after detecting internal standard peaks This ensures that there is no possibility of DNA nicking from the light source Therefore it is critical that the LEDs are calibrated anew before each run The...

Страница 32: ...D Calibration window will launch The Calibration Status field will contain the message Ready for calibration Press CALIBRATE 6 The LED photocurrents will automatically adjust to a factory setting and...

Страница 33: ...l columns Inspect the cassette from the top and underneath Look for any obvious breakage in any of the gel columns 3 Dislodge bubbles from behind the elution modules Hold the cassettes sideways with t...

Страница 34: ...electrode port openings 5 Remove adhesive strips from cassette With the cassette on the benchtop place one hand on the cassette and hold it firmly Grab the white tab of the tape and pull the strip fir...

Страница 35: ...e bottom of the elution modules without sealing the elution port opening If the tips seal the port opening it will be extremely difficult or impossible to empty and refill the elution module completel...

Страница 36: ...erated they will fail the current test until the cassette is at least 17oC 62oF Should this be the case wait until the cassette temperature has equilibrated to room temperature and re test 1 With the...

Страница 37: ...re If these steps do not result in a passed test users should not use the affected lane pairs and contact Sage Science support Failure due to temperature The continuity reading can be affected by temp...

Страница 38: ...the top of the chamber A low level of buffer will have the appearance of an obviously larger bubble when compared to adjacent chambers If this is the case users should add additional buffer until the...

Страница 39: ...d Ficoll as a densifying agent and therefore the samples will sink and form a high density layer beneath the electrophoresis buffer when pipetted slowly into the sample wells If there is insufficient...

Страница 40: ...follow the liquid level up with the tip as the well fills Don t be concerned if the sample well slightly overfills The density of the sample will allow it to sink before it can flow out of the well 1...

Страница 41: ...ing a run Monitoring fluorescent markers in a run Monitoring the progress or status of a run Importing sample identification for a run Operating an optical calibration test Operating a cassette contin...

Страница 42: ...of manufacture are embedded in the serial number Bottom of the cassette This barcode is read by the PippinHT instrument nests The barcode is read when a continuity test is completed and when a run is...

Страница 43: ...s and Shift Tab to scroll up fields Scanned in with a barcode wand a bar code wand may be use with the USB port on the front or back of the PippinHT to scan bar coded sample IDs from a traveler or sam...

Страница 44: ...60005 Rev G 3 In Main screen press IMPORT on the control panel a pop up window will appear 4 In the pop up window select the sample ID file and press IMPORT 5 Press Exit and verify that the informatio...

Страница 45: ...mple ID Template to a size selection protocol This information will prefix the Sample ID in the Main screen 1 In the Protocol Editor when programming a protocol enter a Sample ID Template 2 When the p...

Страница 46: ...ration routine a screenshot of the result is saved as a png file Continuity Test at the end of every continuity test a screenshot of the result is saved as a png file Main Screen at the end of every r...

Страница 47: ...he user the serial number will be in the log file names twice Once the instrument name is modified it will replace the serial number the lower right corner of the Main screen To access the serial numb...

Страница 48: ...in the Protocol Name field in the Run Status screen The protocol that is currently loaded in the Protocol Editor screen will be loaded 4 The different protocol can be loaded by clicking on the Protoc...

Страница 49: ...nual 12 9 460005 Rev G 7 When a run has been started the background color of each running cassette nest will indicate green 8 When a run has been paused the background color of each running cassette n...

Страница 50: ...automatically when every elution is complete 12 4 Monitoring a Run 12 4 1 Run Status Indicators Run status indicators display the following Protocol being run Date and Time Run Progress elapsed time...

Страница 51: ...rs in different states The following information is displayed Nest Number on the screen tab Cassette Serial Number on the screen tab Sample IDs Electrophoresis current lane pair Elution time lane pair...

Страница 52: ...25 and re scans for peaks It will repeat this with an additional 25 decrease in base to threshold if peaks are still not detected If peaks are finally detected the run will proceed as usual If peaks...

Страница 53: ...aks should be called If the run reaches that specified duration and the peaks have not been detected the software decreases the base to threshold value by 25 and re scans for peaks It will repeat this...

Страница 54: ...ct Manual Mode from the Protocol Name drop down menu 2 With a prepared cassette s on the instrument and the lid closed Press START 3 In the lane status indicators lane pair electrophoresis can be star...

Страница 55: ...rmat The Log Review is the third tab 13 1 Overview The Log Review tab is the screen with which users can open saved log files and review the run data that was saved Log Review tab Cassette review tabs...

Страница 56: ...er icon in the Log File window or press LAST to load the file from the latest run on the PippinHT 2 If the folder icon is pressed a pop up window will launch showing a Windows view of file tree The di...

Страница 57: ...ete PippinHT files There are three types of files used by the system log text and image protocol and cassette type The screen is divided into two sub screens the Pippin file directory on the left and...

Страница 58: ...very run or if a run is stopped a text txt file is saved This files contains optical data electrical current data and protocol and sample information from a run Run Log data may be viewed in the Log R...

Страница 59: ...e into the USB port on the front panel of the instrument the USB ports on the back panel may be used as well To transfer from the PippinHT to a USB Drive 1 Select the directory from which the file wil...

Страница 60: ...G To transfer from a USB Drive to the PippinHT 1 Highlight the file to be copied 2 Select the directory from which the file will be copied 3 Press the COPY TO PIPPIN button 4 Press the EJECT FLASH but...

Страница 61: ...he default entry is the instrument serial number Log Prefix This field allows users to enter a string which will be added as a prefix to every log file as it is saved The default is a blank field TCP...

Страница 62: ...e default is a blank field Reversible Field for Sample Recovery When activated all sample collections will terminate with a 5 second field reversal to improve recovery When deactivated all sample coll...

Страница 63: ...oad the Software Upgrade package by pressing the appropriate link on www sagescience com support PippinHT Software Downloads 2 Press Save File when prompted The zip file will likely save to a Download...

Страница 64: ...make sure that only the root directory appears in the sample below E is the root directory of the USB drive 6 The final USB file structure should be as shown The zip file may be deleted 16 2 Upgrading...

Страница 65: ...indow press SOFTWARE UPGRADE 4 Press OK at the warning prompt the Software Upgrade Window will launch 5 The current version of software will be displayed in the left column and the version of software...

Страница 66: ...G 6 When the update is complete the software version numbers will display the same revision number and the UPDATE SYSTEM button will display dark gray 7 Press START PippinHT to return to launch the up...

Страница 67: ...re provided with the PippinHT These should be filled with deionized water and used with the procedure below Important Electrodes should be rinsed after 5 runs or once per week whichever is more freque...

Страница 68: ...y and possibly create a dangerous condition Sage Science support staff may ask for a log file or image file to evaluate or troubleshoot instrument issues High Voltage The PippinHT contains high voltag...

Страница 69: ...oresis Voltage up to 100V constant or pulsed Optical detection 470 nm excitation 525 nm emission Power Requirements 100 240 VAC 10 VAC 2 5 A 50 60 Hz Weight 20 lbs 9 kg Dimensions Closed Position 13 W...

Страница 70: ...pages Reproducibility3 specified on following pages Sample Recovery4 50 80 1 Up to 3 ug of DNA may be loaded however accuracy may be affected by load amounts over 1 5 ug and the distribution profile o...

Страница 71: ...ction Times for Minimum Size Range Tight Targets Target bp Time to Time to Collect min 100 31 150 38 250 47 Run times are affected by temperature and broad range collections Times should be used as an...

Страница 72: ...for Minimum Size Range Tight Targets Target bp Time to Time to Collect min 100 25 200 28 300 32 400 36 500 40 600 45 Run times are affected by temperature and broad range collections Times should be...

Страница 73: ...ion Times for Minimum Size Range Tight Targets Target bp Time to Time to Collect min 500 32 800 37 1100 40 1400 45 Run times are affected by temperature and broad range collections Times should be use...

Страница 74: ...High Pass Marker 15C DNA Marker 15C Typical times to detector internal standards High Pass Threshold bp Time to Time to Collect hr min 500 1 20 1000 1 30 1500 1 40 2000 1 55 Performance Specifications...

Страница 75: ...for Minimum Size Range Tight Targets High Pass Threshold bp Time to Time to Collect min 6000 63 7000 67 8000 70 9000 74 Run times are affected by ambient temperature Times should be used as an approx...

Страница 76: ...for Minimum Size Range Tight Targets High Pass Threshold bp Time to Time to Collect hr min 15000 2 20 17000 2 34 20000 2 45 Run times are affected by ambient temperature Times should be used as an app...

Страница 77: ...for Minimum Size Range Tight Targets High Pass Threshold bp Time to Time to Collect hr min 30000 7 00 35000 8 30 40000 9 00 Run times are affected by ambient temperature Times should be used as an app...

Страница 78: ...PippinHT Operations Manual 20 9 460005 Rev G 500 Cummings Center Suite 2400 Beverly MA 01915...

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