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Sage Science BluePippin Operation Manual Download Page 24

      978-922-1832      

●

            www.sagescience.com        

●

      [email protected] 

 
 

BluePippin Operations Manual 460013 Rev F                         

                           7-4 

 

 

7.3    Selecting a Cassette Definition 

 

Cassettes are selected from a menu tree with the 2 tiers.  Users should select the 
appropriate definition from the 1

st

 or 2

nd

 tier: 

 

•

 

1

st

 Tier: % Agarose

 

of the cassette

 

▪

 

3%

 

▪

 

2%

 

▪

 

1.5%

 

▪

 

0.75%

 

 

•

 

2

nd

 Tier: Additional cassette definitions 

If a cassette type (e.g. 0.75% Agarose, illustrated below) has more than one 
definition, the definition is selected from the second tier of the menu tree. 

 

▪

 

1 

– 6 kb Low Voltage  

▪

 

1 

– 6 kb Pulsed Field 

▪

 

3 

– 10 kb Pulsed Field 

▪

 

18 

– 27 kb Pulsed Field 

▪

 

50 kb Pulsed Field 

 

 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

 

Summary of Contents for BluePippin

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

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

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