DNA Size Selection System
Operations Manual
Страница 1: ...DNA Size Selection System Operations Manual...
Страница 2: ...Suite 2400 500 Cummings Center Beverly MA 01915 2018 Sage Science Inc All rights reserved Sage Science and BluePippin are trademarks of Sage Science Inc All other brands and name mentioned herein are...
Страница 3: ...S 3 4 4 UNPACKING AND INSTALLATION 4 1 5 WORKFLOW SUMMARY 5 1 6 SAMPLE PREPARATION 6 1 7 PROGRAMMING GUIDE 7 1 7 1 Overview 7 1 7 2 Programming Summary 7 2 7 3 Selecting a Cassette Definition 7 4 7 4...
Страница 4: ...Y AND YIELD 14 1 14 1 Overview 14 1 14 2 Intrinsic Sample Recovery on the BluePippin 14 1 14 3 Improving Product Yield by Selecting a Wider Size Range 14 2 14 4 Improving Product Recovery with the Fie...
Страница 5: ...4 25 4 1 5 Agarose 250 bp 1 5 kb Marker R2 internal standards 25 5 25 5 1 5 Agarose High Pass Marker R2 internal standards 25 6 25 6 0 75 Agarose 2 6 kb Marker S1 Low Voltage external marker 25 7 25...
Страница 6: ...keyboard The electrode array is located in the top of the sliding instrument cover and is not user accessible Software System software allows the user to enter parameters that define the DNA fragment...
Страница 7: ...ting dyes or gel stains sample DNA is not viewable or monitored Size Selections can be undertaken using one of the following strategies Labeled internal standards Fluorescein labeled DNA internal stan...
Страница 8: ...Range setting The cassette lane will collect a distribution of fragments between a starting base pair value and an ending base pair value The actual start and end fragment sizes are typically within 1...
Страница 9: ...ration of the cassettes Section 10 Sample loading Section 11 2 1 Common Misconceptions Users should be aware of the following characteristics of the BluePippin System These are part of normal operatio...
Страница 10: ...properly Figure 2 2 An illustration 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...
Страница 11: ...oving Sample Yield for HMW DNA Recovering high molecular weight DNA 2 kb using all 0 75 agarose gel cassettes tend to have lower yields than low molecular weight size selection The following additiona...
Страница 12: ...Important Provide important information about the proper use of the system that may influence the quality of the result Information Provides additional information regarding the function of the syste...
Страница 13: ...cond box will contain the computer monitor in the manufacturer s original packaging With the boxes in the upright position open and confirm that the following items are enclosed Monitor LCD computer m...
Страница 14: ...onnect monitor to power using power supply supplied with monitor 5 Insert USB connector from computer keyboard into port located in the back of the Instrument 6 Connect mouse to BluePippin via USB or...
Страница 15: ...support sagescience com BluePippin Operations Manual 460013 Rev F 4 3 Power Entry Port Power Switch Figure 4 1 Rear Panel Ports Figure 4 2 Rear Panel Connections USB Ports 4 VGA monitor port Power Cab...
Страница 16: ...m BluePippin Operations Manual 460013 Rev F 4 4 Figure 4 3 BluePippin front panel USB Port Indicator Light Blue when instrument is ready for use power is on and software is active Indicator Light Gree...
Страница 17: ...r 10 cassettes store at 4 o C 550 l DNA internal standards loading solution mix 10 l sample 40 ml of spare running buffer Dye free gels with labeled external marker 40 samples size selections 2kb o 10...
Страница 18: ...rage requirements should be observed Gel Cassettes Store at room temperature in the foil bags until ready for use Internal standards or external markers store at 4o C minimize time at room temperature...
Страница 19: ...standard HMW protocol 2kb 0 75 agarose Yes No Prepare samples add loading solution to samples use external marker max 4 samples cassette external marker Program a Size Selection Protocol Select USE IN...
Страница 20: ...brane at the back of the elution module For best results samples should be de proteinized prior to loading whenever possible Input DNA size distribution A knowledge of the input size distribution is o...
Страница 21: ...users create new protocols or edit existing protocols 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...
Страница 22: ...nd different modes 7 2 Programming Summary The following is a summary of the steps required to program a protocol Detailed instructions are provided in the subsequent sections 1 Click the Protocol Edi...
Страница 23: ...e size selection protocol and assign the DNA reference marker lane There are three modes of programming on the BluePippin Tight collects minimum allowable distribution range of DNA fragments using the...
Страница 24: ...should select the appropriate definition from the 1st or 2nd tier 1st Tier Agarose of the cassette 3 2 1 5 0 75 2nd Tier Additional cassette definitions If a cassette type e g 0 75 Agarose illustrated...
Страница 25: ...default settings The 8 hour default value in the Run Time field ensures that a run is not unintentionally ended early The checked box in the End Run when Elution is Completed field will automatically...
Страница 26: ...2 In the protocol editor enter the reference lane number into the Reference Lane field Click APPLY REFERENCE TO ALL LANES Figure 7 1 shows the proper settings for running a DNA marker in lane 1 with p...
Страница 27: ...to the sample The software will determine the timing of elution based on the migration of the DNA marker within its own lane 3 Figure 7 2 shows the correct assignation of marker lanes for internal st...
Страница 28: ...larger than the minimum has been selected for a sample lane the BP Range Flag field for that sample will display broad and an orange color Narrow Range Flag If the Range collection mode is selected an...
Страница 29: ...v F 7 9 Pause Enabled Indicator If the pause feature has been entered for a sample lane Pause On indicator will be displayed Warnings Text Box If there are errors or exceptions within a programmed pro...
Страница 30: ...un and external reference marker Assignation of reference DNA is detailed in Sections 7 5 and 7 6 In the protocol editor 1 Select an appropriate cassette type for size selection if a new protocol is b...
Страница 31: ...a starting and ending base pair value in software The system will collect the fragment range between the start and end base pair values This option should be used when collections that are broader th...
Страница 32: ...the instrument run to collect additional sample This will result in the collection of two contiguously sized fractions with overlapping fragment sizes Two collections of a paused Tight setting not rec...
Страница 33: ...and T End The pause function temporarily turns off power to the electrodes and suspends run timers allowing users to remove the sample rinse the elution well if desired and then resume the instrument...
Страница 34: ...The BluePippin optical system must be calibrated before every run The instrument is supplied with a calibration fixture that fits over the LED detectors on the optical nest 1 Press the Main Tab 2 Pre...
Страница 35: ...2 3 An LED Calibration window will launch The Calibration Status field will contain the message Ready for Calibration Place fixture on the optical nest so that all five LED detectors are covered The d...
Страница 36: ...978 922 1832 www sagescience com support sagescience com BluePippin Operations Manual 460013 Rev F 9 3 4 Close the lid 5 Press CALIBRATE...
Страница 37: ...The Calibration status will read Calibration Underway and the LED photocurrents will automatically adjust to a factory setting Once calibration is successful the Calibration Status field will contain...
Страница 38: ...m edges Reservoirs should be nearly full of buffer and roughly equal in volume across the cassette If the buffer level in any reservoir appears less than 50 full compared with its neighbors the low re...
Страница 39: ...fected since the DNA travels through the center of the gel column above the bubble To find such bubbles turn the cassette upside down and view the bottom of the channels under a strong light while til...
Страница 40: ...in the elution reservoirs won t be trapped behind the elution modules Important Be sure the cassette is fully seated into the bottom of the nest Detection of DNA within the cassette will fail if the...
Страница 41: ...tape over the elution wells and rub firmly to fix the tape in position For best results rub the tape at the boundary of the well with a smooth hard object such as the back end of a lab marker pen 6 C...
Страница 42: ...is highlighted in orange Figure 10 5 shows a failed test screen elution channel in lane 1 2 If a Separation lane has failed left column continuity Do not use that lane Remaining passing lanes can be u...
Страница 43: ...ll as a densifying agent see the previous chapter on Sample Preparation for details and therefore the samples will sink and form a high density layer beneath the electrophoresis buffer when pipetted s...
Страница 44: ...ample well In addition there is an agarose chimney surrounding the top of the sample well that protrudes up through the cassette cover see Figure 11 1 When removing buffer some users find it useful to...
Страница 45: ...fault screen on the BluePippin is a tabbed format The Main Tab is the first tab and the default screen 12 1 Overview The Main Tab is the screen with which users load protocols run the instrument and m...
Страница 46: ...e Protocol Status Panel The last protocol that has been programmed will automatically be loaded See Chapters 7 and 8 for instructions on programming 2 Load a properly prepared cassette Chapter 10 into...
Страница 47: ...t gray When a protocol is running the following indicator panel will be displayed Green Separation If a lane is separating fragments the Separate indicator will be green Orange Elution If a lane is el...
Страница 48: ...ed The elution timer will show the value of the total elapsed elution time Run progress is monitored in the Protocol Status panel The percent completion of the run time is indicated by a progress bar...
Страница 49: ...calibration tests are also automatically saved All files may be accessed from the File Manager Tab The files are saved internally on the Pippin hard drive in the directory named home pippin BluePippi...
Страница 50: ...mples from standard cassettes with sealed elution modules will be recovered in approximately 40 l of electrophoresis buffer 4 When all samples are collected remove used cassette from instrument and di...
Страница 51: ...pin automatically adds a brief field reversal at the end of each elution to push DNA off of the membrane Section 14 4 However for higher molecular weight DNA collections 2kb we recommend several user...
Страница 52: ...the input DNA will be recovered Users should broaden their collection ranges if the default tight settings do not produce enough DNA for their application Figure 14 2 shows an example of the effect o...
Страница 53: ...but may be turned off by users Figure 14 3 below shows the improvement that can be achieved Figure 14 4 shows the setting option in software When activated the setting will be applied to all subseque...
Страница 54: ...re removing eluted DNA it is OK to leave samples in instrument for hours i e for an overnight run 2 Additional 10 30 yield can often be achieved by rinsing the elution well with Tween 20 0 1 Tween 20...
Страница 55: ...DNA from E coli For each cassette type a different restriction digest is used chosen so that size distribution of the digested DNA closely matches the useful fractionation range of the cassette withou...
Страница 56: ...er UDP Server Port Provides an address for remote communications between the BluePippin and wireless control Contact Sage Science for support Reversible Field for Sample Recovery When activated all sa...
Страница 57: ...eld for Sample Recovery button The indicator display will change from dark gray to white and the display will change from OFF to ON 2 The Settings Changed indicator light will activate Press Accept to...
Страница 58: ...control 1 Select the Manual Mode from the top of the Protocol Name drop down menu in the Run File Manager 2 Press Start on the controller 3 Sample lanes may be run individually by pressing the Separat...
Страница 59: ...Pause Resume and Stop buttons are also operative as in automated runs Note There is a delay between the time that a fragment is detected and visible on the graph or image and when it is in position to...
Страница 60: ...ize selection runs All programming parameters are displayed as originally entered in the Protocol Editor Optical and electrical current data from the run are displayed in graphical windows Specific re...
Страница 61: ...3 Rev F 18 2 Opening a Log file in the Review Screen Note A stand alone Review Log Program is available for PCs Contact sage science support i Click the Log Review tab along top of the screen ii To se...
Страница 62: ...Current graph A check mark next to each lane will toggle the visualization of that lane on and off The type and color of the lines may also be adjusted by clicking inside the line field and holding th...
Страница 63: ...at are listed in the DNA marker times and values list The yellow vertical line indicates the estimated base pair position at a given run time The indicator may be moved see below along the x axis show...
Страница 64: ...results in mA The timing of an elution switch from separation to elution is accompanied by a 0 5 mA drop in current There are three icons in the upper right hand corner of the graph The functions are...
Страница 65: ...File Manager Tab allows users to access copy and delete BluePippin 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...
Страница 66: ...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 Review screen Se...
Страница 67: ...sfer a file place a USB drive 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 BluePippin to a USB Drive 1 Select the dir...
Страница 68: ...ions Manual 460013 Rev F 19 4 To transfer from a USB Drive to the BluePippin 1 Highlight the file to be copied 2 Select the directory from which the file will be copied 3 Press the COPY TO PIPPIN butt...
Страница 69: ...20 1 Extracting the Files to a USB flash drive 1 Download the Software Upgrade package by pressing the appropriate link on www sagescience com support in the Downloads section under Software 2 Press S...
Страница 70: ...unch to prompt you to select a destination where to extract files The prompt will suggestcreating folder based on the zip file name Delete the destination folder name so that only the root directory a...
Страница 71: ...pin Instrument Software 1 Insert the USB drive with the upgrade folder into the USB port on the front panel of Pippin instrument 2 From the Main Tab press INFO This will launch the information window...
Страница 72: ...ive prior to removal Failure to do so may cause data loss and problems with the upgrade procedure 7 Press EXIT to return to Main Menu This will install the software Installation may take up to 30 seco...
Страница 73: ...ette is provided with the BluePippin This should be used with the procedure below No other preventative maintenance is required Important Electrodes should be rinsed after 5 runs or once per week whic...
Страница 74: ...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 BluePippin contains high volt...
Страница 75: ...s Internal Standards Catalogue No 3 agarose dye free 100 250 bp Q3 BDF3010 2 agarose dye free 100 600 bp V1 BDF2010 1 5 agarose dye free 250 1500 bp R2 BDF1510 1 5 agarose dye free High Pass 250 R2 BD...
Страница 76: ...Ext Mkr 10 27 kb 5 g load 16 loads CON7604 Control DNA for 0 75 Gel Cassette Ext Mkr High Pass 5 g load 16 loads CHP7510 Reagent Replacement Kits Dye Free with Internal Standards Catalogue No BDF3010...
Страница 77: ...tions Specifications for BluePippin Instrument Electrophoresis Voltage 25V 100V 150V constant 100 V pulsed Optical detection 470 nm excitation 525 nm emission Power Requirements 100 240 VAC 10 VAC 2 5...
Страница 78: ...mple Recovery4 50 80 1 Amounts DNA above the maximum may be loaded however accuracy may be affected DNA will migrate more slowly 2 Deviation of actual target value from software input value divided by...
Страница 79: ...tor Estimated Collection Times for Minimum Size Range Tight Targets Target bp Time to Time to Collect min 100 65 140 70 180 80 240 90 Run times are affected by temperature and broad range collections...
Страница 80: ...imated Collection Times for Minimum Size Range Tight Targets Target bp Time to Time to Collect min 100 49 200 57 300 63 400 71 500 79 600 86 Run times are affected by temperature and broad range colle...
Страница 81: ...stimated Collection Times for Minimum Size Range Tight Targets Target bp Time to Time to Collect min 150 52 275 63 400 70 500 76 600 82 Run times are affected by temperature and broad range collection...
Страница 82: ...es for Minimum Size Range Tight Targets Target bp Time to Time to Collect min 250 29 300 30 600 37 1200 49 1500 56 Run times are affected by temperature and broad range collections Times should be use...
Страница 83: ...ion 1 5 DF Marker R2 250 1500bp high pass DNA Marker R1 Typical times to detector Performance Specifications Accuracy BPStart 10 Reproducibility BPStart 10 Specifications subject to change without not...
Страница 84: ...cal times to detector Estimated Collection Times for Minimum Size Range Tight Targets Run times are affected by temperature and broad range collections Times should be used as an approximate guideline...
Страница 85: ...Typical times to detector Estimated Collection Times for Minimum Size Range Tight Targets Run times are affected by temperature and broad range collections Times should be used as an approximate guid...
Страница 86: ...S1 Typical times to detector Estimated Collection Times for Minimum Size Range Tight Targets Target bp Time to Time to Collect hr min 5 000 2 23 6 000 2 32 8 000 2 51 10 000 3 10 Run times are affecte...
Страница 87: ...r S1 High Pass 15 20kb DNA Marker S1 Typical times to detector Estimated Collection Times 4 10kb high pass 2 hours 6 10kb high pass 2 hours 15 20kb high pass 5 6 hours Performance Specifications Versi...
Страница 88: ...n Version 3 0 75 DF Marker U1 high pass 30 40kb vs3 DNA Marker U1 Typical times to detector Approximate Collection Time is 9 5 10 hours Performance Specifications Accuracy BPStart 10 Reproducibility B...
Страница 89: ...ical times to detector Estimated Collection Times for Minimum Size Range Tight Targets Target bp Time to Time to Collect hr min 10 000 2 55 12 000 3 00 15 000 3 15 17 000 3 25 18 000 3 35 Run times ar...
Страница 90: ...arker T1 Typical times to detector Estimated Collection Times for Minimum Size Range Tight Targets Target bp Time to Time to Collect hr min 18 000 3 45 23 000 4 00 27 000 4 15 Run times are affected b...
Страница 91: ...ctor Estimated Collection Times Target hr min Add min 50kb tight 7 50 15 50kb broad 8 50 30 Target 50kb tight 50kb broad Minimum Size Distribution as CV 10 14 Accuracy 10 10 Reproducibility 10 10 Spec...
Страница 92: ...978 922 1832 www sagescience com support sagescience com BluePippin Operations Manual 460013 Rev F 25 15 500 Cummings Center Suite 2400 Beverly MA 01915...
