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Sage Science PippinHT Operation Manual

Dna size selection system
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DNA Size Selection System
Operations Manual

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  • Page 1 DNA Size Selection System Operations Manual...
  • Page 2 PippinHT trademarks of Sage Science, Inc. All other brands and name mentioned herein are property of their owners. The PippinHT instrument and gel cassettes are covered by U.S. patents 8,361,298 and 8,361,299. For research use only. PippinHT Operations Manual 460005 Rev G...

  • Page 3: Table Of Contents

    SAMPLE PREPARATION ....................6-1 6.1 Input Sample Characteristics ....................6-1 6.2 Preparing DNA Samples for the PippinHT ................6-1 6.2.1 Sample preparation for use with an internal standard ............6-1 6.2.2 Sample preparation for use with an external marker ............6-2 6.3 Recommended sample Load Guidelines .................
  • Page 4 LOG REVIEW ......................13-1 13.1 Overview ..........................13-1 13.2 Loading a file ........................13-2 MANAGING FILES ....................14-1 14.1 Overview ..........................14-1 14.2 File Types .......................... 14-2 14.3 Transferring files ....................... 14-3 SYSTEM OPTIONS....................15-1 PippinHT Operations Manual 460005 Rev G...
  • Page 5 ● ● CONTENTS (Cont'd) UPGRADING PIPPINHT SOFTWARE ............... 16-3 16.1 Extracting the Files to a USB flash drive............... 16-3 16.2 Upgrading the Pippin Instrument Software..............16-4 PREVENTATIVE MAINTENANCE – RINSING ELECTRODES ......... 17-7 17.1 Electrode Rinse Procedure ....................17-7 WARRANTY AND SERVICE ..................18-1 INSTRUMENT SPECIFICATIONS ................

  • Page 6: Introduction

    ● ● Introduction Thank you for purchasing the PippinHT from Sage Science. The PippinHT is an automated preparative gel electrophoresis system. We urge you to read this manual to familiarize yourself with the system’s capabilities and precautions. 1.1 System Overview There are three components to the PippinHT system: •...

  • Page 7: How The System Works

    Indicator lights Barcode readers USB Port Figure 1.1. Layout of the PippinHT instrument 1.2 How the system works The schematic in Figure 1.2 Illustrates agarose gel columns during separation of DNA fragments. Note that the optical detectors monitor the odd numbered lanes only.

  • Page 8: Collection Strategies

    Up to 12 samples may be run per cassette. Note: The DNA sequence of internal standards may be found at www.sagescience.com/support on the PippinHT resources page, under the Quick Guide download section. PippinHT Operations Manual 460005 Rev G...

  • Page 9: Collection Types

    Timed runs – The beginning and end time of elution is set by the user. Neither internal standards or external markers are used. 1.4 Collection Types The PippinHT can be programmed to size select either targeted fractions or “High-Pass” collections. The fragment size distributions are described below: 1.

  • Page 10: Keys To Successful Collections

    2.3, below. 2.1 Common Misconceptions Users should be aware of the following characteristics of the PippinHT system. These are part of normal operation of the system, but may seem counterintuitive at first. 2.1.1 Narrowest is not always the best.
  • Page 11 600 bp. The collections of large DNA fragments, especially with pulsed –field protocols, can also appear counter-intuitive with respect to timing. Figure 2.3. An illustration comparing the relative rate of electrophoresis in the separation and elution channels PippinHT Operations Manual 460005 Rev G...

  • Page 12: Improving Sample Yield

    Yield can be affected by DNA becoming lightly bound to the low molecular weight cut- off membrane in the elution well. At the end of the run, the PippinHT will briefly reverse field (for 5 seconds) in every elution channel. This improves sample yield by up to 20%.

  • Page 13: Safety And Precautions

    Wear a lab coat, safety glasses, and gloves. • Use in proximity of an eye wash station and/or running water. Caution! The PippinHT was designed to be operated on a flat surface. Do not operate on a tilted surface or tilt during operation. PippinHT Operations Manual...

  • Page 14: Unpacking And Installation

    2. Remove the cardboard shelf from the box. 3. Remove the foam insert at the top of the instrument. 4. Reach into the box and grab the instrument from the bottom. The PippinHT weighs approximately 20 lbs.
  • Page 15 14. Press power switch located on the rear of the instrument. The blue light on the front panel will turn on (figure 4.2). 15. Wait for software to launch, this will take 30 seconds - 1 minute. The PippinHT is ready for use.

  • Page 16: Unpacking Gel Cassette Kits

    4 X 800l of 0.1% Tween20 in running buffer. Tween solution is used to improve recovery of large DNA fragments. Important! Fluorescein labels will degrade at room temperature – minimize time at RT for labeled standards and markers PippinHT Operations Manual 460005 Rev G...

  • Page 17: Storage Conditions For Cassettes And Reagents

    Return to 4ºC, immediately after use. • Electrophoresis buffer – may be stored at room temperature or 4ºC. Equilibrate to room temperature before use. • Loading solution – store at 4ºC. Equilibrate to room temperature before use. PippinHT Operations Manual 460005 Rev G...

  • Page 18: Workflow Summary

    Enter reference lane No. for the external marker Select “APPLY TO ALL LANES” Calibrate the Optics Prepare the Cassette(s) Run the Continuity Test on the Cassette(s) Load Samples onto the Cassette(s) Run the PippinHT PippinHT Operations Manual 460005 Rev G...

  • Page 19: Sample Preparation

    ● ● Sample Preparation 6.1 Input Sample Characteristics When running the PippinHT™, characteristics of input DNA can affect separation resolution and efficiency of product recovery. The following general guidelines should be followed: • Ionic strength: The ionic strength of the sample should be lower than the ionic strength of the buffer (80mM monovalent ions).

  • Page 20: Sample Preparation For Use With An External Marker

    B. For 0.75% agarose gel cassettes (High-Pass size selection) 1. Bring the external marker solution to room temperature. 6.3 Recommended sample Load Guidelines Maximum Load: 1.5 g sheared genomic DNA Minimum Load: 15 ng sheared genomic DNA PippinHT Operations Manual 460005 Rev G...

  • Page 21: Programming A Protocol

    ● ● Programming a Protocol The default screen on the PippinHT has a tabbed format. The Protocol Editor is the second tab. 7.1 Overview The Protocol Editor tab is the screen with which 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 “.phprot”...

  • Page 22: Protocol Parameters Detail

    Note: Range mode is used for programming High-Pass protocols. – • Time collects fragments base on a starting elution time (time after the beginning of a run) and ending elution time. ote: Programming modes may be independently applied to individual sample lanes PippinHT Operations Manual 460005 Rev G...
  • Page 23 “narrow”. These collections will not have a narrower fragment distribution, but the specified minimum with a lower yield: Pause On The Pause On displays a light gray color if the pause feature has been enabled for that lane-pair. PippinHT Operations Manual 460005 Rev G...

  • Page 24: Writing A Size Selection Protocol

    It is important to keep track of which cassette is being programmed. Note: Parameters programmed on Cassette 1 may be copied to Cassette 2, but not vice versa. If creating a 2-cassette protocol, starting with cassette 1 is recommended. PippinHT Operations Manual 460005 Rev G...
  • Page 25 This does not have a bearing on the actual run time, which ends when the final elution is complete. Users should not change this value or uncheck the box in most run scenarios. PippinHT Operations Manual 460005 Rev G...
  • Page 26 (hh:mm:ss) values only (a reference DNA marker is not used) 6. Enter Sample ID Template information (optional). These will be saved with the protocol and prefix to Sample ID information imported (also optionally) into the Main screen when initiating a run. PippinHT Operations Manual 460005 Rev G...
  • Page 27 7. 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 may be copied, however. COPY LANE 1 TO CASSETTE PippinHT Operations Manual 460005 Rev G...
  • Page 28 Warning! The copy functions will overwrite any pre-entered values. 11. Press SAVE AS to save the protocol. A pop-up window will appear that displays the protocols folder. Enter a file name and press OK. PippinHT Operations Manual 460005 Rev G...

  • Page 29: Using An External Marker

    3. Press the APPLY TO ALL LANES button to assign size selection in remaining lanes based on the external marker designated lane. The reference lane should appear in every “Ref Lane” field when using an external marker PippinHT Operations Manual 460005 Rev G...
  • Page 30 Start value (not the Target value) is higher than any other Start value in the remaining lanes. Users should be very careful when doing this. 5. Refer to back to Section 7.2 for the remaining instruction for programming a protocol. 7-10 PippinHT Operations Manual 460005 Rev G...

  • Page 31: Optical Calibration

    ● Optical Calibration The PippinHT optical system must be calibrated before every run. The instrument is supplied with two calibration fixtures that fit over the LED detectors on the optical nest. Important! The LED optical array turns off after detecting internal standard peaks. This ensures that there is no possibility of DNA nicking from the light source.
  • Page 32 “Ready for calibration”. Press CALIBRATE. 6. The LED photocurrents will automatically adjust to a factory setting, and the “Calibration Status” field will contain the message “Calibration OK”. 7. Press EXIT to return to the Main Tab. PippinHT Operations Manual 460005 Rev G...

  • Page 33: Preparing A Cassette

    Use the schematic below as a guide. Tap the side of the cassette Dislodge bubbles upwards from behind the elution wells PippinHT Operations Manual 460005 Rev G...
  • Page 34 6. Remove buffer from the upper electrode buffer chambers For 3%, 2% or 1.5% gel cassettes, remove 80 μl. For 0.75% gel cassettes, remove 150 μl . Remove buffer PippinHT Operations Manual 460005 Rev G...
  • Page 35 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. 9. Place the cassette(s) onto the PippinHT nest(s) PippinHT Operations Manual 460005 Rev G...

  • Page 36: Continuity Test

    3. Continuity Test will Automatically Run. The continuity test pop- screen will launch. The separation and elution channel test parameters are displayed and the electrical current results will be listed after several seconds. 10-1 PippinHT Operations Manual 460005 Rev G...

  • Page 37: Continuity Test Failure And Troubleshooting

    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 temperature. If this is the case, the continuity reading will only be slightly different than the other lane-pairs (or they will all fail, but have similar values).
  • Page 38 Figure 10.1. Failed Separation continuity test, lane-pair 3/4. Check upper and lower buffer chamber buffer levels. Figure 10.2. Failed Elution continuity test, lane-pair 3/4. Check upper and middle buffer chamber buffer levels, and replace elution well buffer. 10-3 PippinHT Operations Manual 460005 Rev G...

  • Page 39: Loading Samples

    Loading Samples 11.1 Sample loading best practices Proper sample loading is critical for best performance of PippinHT cassettes. For maximum reproducibility and accuracy, the sample should travel through the central section of the gel column, and should be bounded on all four sides by uniformly conductive media –...

  • Page 40: Sample Loading Procedure

    11.3 Loading less than 12 samples, and reusing a gel cassette It is possible to run less than 12 samples on a PippinHT gel cassette. When a size selection protocol is programmed, only the lane-pairs which have been assigned a function (size selection or external marker) will be used.

  • Page 41: Running A Protocol

    ● ● Running a Protocol The default screen on the PippinHT is a tabbed format. The Main screen is the first tab. 12.1 Overview The Main screen is the interface with which users control the PippinHT instrument and initiate operations for running gel cassettes. Its functions include: •...

  • Page 42: Sample Tracking

    • 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 started. The barcode text is displayed in the nest tabs on the Main screen and recorded to the log file.

  • Page 43: Sample Id Entry

    12.2.2 Sample ID entry Sample ID information may be entered in the Sample ID field for both cassettes that are run on the PippinHT. Information may be entered in any of these ways: • Keyed in manually – use the Tab key scroll down fields and Shift-Tab to scroll up fields.
  • Page 44 In Main screen, press IMPORT on the control panel, a pop-up window will appear. In the pop-up window, select the sample ID file and press IMPORT. Highlight file Press Exit and verify that the information has been imported. 12-4 PippinHT Operations Manual 460005 Rev G...

  • Page 45: Using The Sample Id Template

    1. In the Protocol Editor when programming a protocol, enter a Sample ID Template. 2. When the protocol is saved or loaded, the Sample ID Template information will appear in the Sample ID fields in the Main Screen. 12-5 PippinHT Operations Manual 460005 Rev G...

  • Page 46: Log Files

    3. When sample IDs are imported (see the previous section 12.2.3), the text fields will concatenate with the Sample ID Template as a prefix. 12.2.5 Log Files Several log files are automatically saved after key operations on the PippinHT: • Optical Calibration -- at the end of every optical calibration routine, a screenshot of the result is saved as a .png file.

  • Page 47: Log File Name Format

    Main screen. To access the serial number from software, click the PippinHT logo to launch the Info pop-up screen. The serial number (Instrument ID) is displayed on the right side. Click the pop-up to return to the main menu.

  • Page 48: Starting, Pausing And Run Completion

    To begin a run, Press the START in the control panel. A pop-up window will appear to remind users that the LED calibration and continuity tests were not inadvertently skipped. Press “OK” to start the run. 12-8 PippinHT Operations Manual 460005 Rev G...
  • Page 49 ● When a run has been started the background color of each running cassette/nest will indicate green: When a run has been paused the background color of each running cassette/nest will indicate blue: 12-9 PippinHT Operations Manual 460005 Rev G...

  • Page 50: Monitoring A Run

    • Lid Status Note: The progress bar and % run value are based on the preset maximum run-time (displayed in the Protocol Editor screen), and has no bearing on the actual run times. 12-10 PippinHT Operations Manual 460005 Rev G...

  • Page 51: Lane Status Indicators

    • Lane-pair is idle (light gray, inactive or run complete) • Lane-pair is separating (green) • Lane-pair is eluting (orange) Lane-pair is inactive Lane-pair is separating Lane-pair is eluting Lane-pair run is complete 12-11 PippinHT Operations Manual 460005 Rev G...

  • Page 52: Marker Detection Failure Alert

    ● 12.4.3 Marker Detection Failure Alert The PippinHT has will run a fail-safe routine if a marker is not detected within the expected time window. Each cassette definition includes a time value by within which all peaks should be called.
  • Page 53 'idle'. This will prevent sample loss. All others lanes will proceed with the run. Users can then choose to manually elute their sample from that lane once the run is complete (see next section, 12.5 for the manual procedure). 12-13 PippinHT Operations Manual 460005 Rev G...

  • Page 54: Manual Mode

    ● ● 12.5 Manual Mode The PippinHT may be run manually without a programmed protocol. 1. In the run status indicator, select “Manual Mode” from the Protocol Name drop-down menu: 2. With a prepared cassette(s) on the instrument, and the lid closed, Press START.

  • Page 55: Log Review

    ● ● Log Review The default screen on the PippinHT has a tabbed format. 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.

  • Page 56: Loading A File

    1. Press the folder 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.

  • Page 57: Managing Files

    ● ● Managing Files The default screen on the PippinHT is a tabbed format. The File Manager Tab is the fourth tab. 14.1 Overview The File Manager Tab allows users to access, copy, and delete PippinHT files. There are three types of files used by the system; log (text and image), protocol and cassette type.

  • Page 58: File Types

    PippinHT during manufacture or through software upgrades. Note. The PippinHT is equipped with 25 GB of storage. The combined files sizes for the log files saved for each run is about 150 kb, or a total storage capacity for 166,000 runs.

  • Page 59: Transferring Files

    14.3 Transferring files Files may be copied between the PippinHT and a USB flash drive. To transfer 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).
  • Page 60 ● ● 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” button.

  • Page 61: System Options

    ● ● System Options The default screen on the PippinHT is a tabbed format. The System Options Tab is the fifth tab. System Log Prefix Options Server Port Instrument Date and Name Time Sample ID Sample ID Prefix Folder in LIMS...
  • Page 62 When activated, the current will be reversed on all subsequent runs until the user changes the setting through the System Options tab. Note: Users should contact Sage Science for detailed protocols for group control of PippinHTs and/or LIMS integration.

  • Page 63: Upgrading Pippinht Software

    ● ● Upgrading PippinHT Software Pippin upgrade software is available for download at www.sagescience.com/support/. The files are provided in a zip file, and the contents must be extracted to the root directory of a USB flash drive. 16.1 Extracting the Files to a USB flash drive Download the Software Upgrade package by pressing the appropriate link on (PippinHT “Software Downloads”).

  • Page 64: Upgrading The Pippin Instrument Software

    Upgrading the Pippin Instrument Software Insert the USB drive with the upgrade folder into the USB port on the front panel of PippinHT instrument. From the Main Tab, click on the PippinHT logo. This will launch the information window. 16-4...
  • Page 65 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 to be installed will display in the right column. Press “UPDATE SYSTEM”. 16-5 PippinHT Operations Manual 460005 Rev G...
  • Page 66 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 updated software and return to the Main Screen.

  • Page 67: Preventative Maintenance - Rinsing Electrodes

    Preventative Maintenance – Rinsing Electrodes It is possible that salts will accumulate on the electrodes that are housed in the PippinHT lid assembly. This may alter the performance of the instrument over time. Two empty cassette bodies are provided with the PippinHT. These should be filled with deionized water and used with the procedure below.

  • Page 68: Warranty And Service

    Do not attempt to repair or disassemble the PippinHT instrument. This will void the remaining warranty 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.

  • Page 69: Instrument Specifications

    ● ● Instrument Specifications Specifications for PippinHT Instrument Electrophoresis 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 •...

  • Page 70: Gel Cassettes Specifications

    2. Deviation of actual target value from software input value divided by the actual value. 3. 2X standard deviation of replicate samples. 4. Recovery is measured using known amounts of plasmid marker ladder. 20-1 PippinHT Operations Manual 460005 Rev G...

  • Page 71: 3% Agarose, 100 Bp-250 Bp, Marker 30G

    Performance Specifications* 100-250 bp Minimum Size Distribution as CV <5% 50 – 80% Sample Recovery Accuracy <5% Reproducibility < 5% Specifications subject to change without notice * see page 20-1 for specification definitions 20-2 PippinHT Operations Manual 460005 Rev G...

  • Page 72: 2% Agarose, 100 Bp-600 Bp, Marker 20B

    Performance Specifications* 100-600 bp Minimum Size Distribution as CV <8% 50 – 80% Sample Recovery Accuracy <5% Reproducibility < 5% Specifications subject to change without notice * see page 20-1 for specification definitions 20-3 PippinHT Operations Manual 460005 Rev G...

  • Page 73: Agarose, 250 Bp-1.5 Kb, Marker 15C

    Performance Specifications* 250-1500 bp Minimum Size Distribution as CV <8% 50 – 80% Sample Recovery Accuracy <5% Reproducibility < 5% Specifications subject to change without notice * see page 20-1 for specification definitions 20-4 PippinHT Operations Manual 460005 Rev G...

  • Page 74: Agarose, High-Pass, >300Bp-1.5Kb, Marker 15C

    1000 1:40 1500 1:55 2000 Collects fragments up to 5- 8kb Performance Specifications* Accuracy, “BPStart” <10% Reproducibility, “BPStart” < 10% Specifications subject to change without notice * see page 25-1 for specification definitions 20-5 PippinHT Operations Manual 460005 Rev G...

  • Page 75: Agarose, High-Pass >6-10 Kb, Marker 75D

    >6000-10000 bp Minimum Size Distribution as CV 50 – 80% Sample Recovery Start Value Accuracy <10% Start Value Reproducibility < 10% Specifications subject to change without notice * see page 20-1 for specification definitions 20-6 PippinHT Operations Manual 460005 Rev G...

  • Page 76: Agarose, High-Pass >15-20 Kb, Marker 75E

    >15000-20000 bp Minimum Size Distribution as CV 50 – 80% Sample Recovery Start Value Accuracy <10% Start Value Reproducibility < 10% Specifications subject to change without notice * see page 20-1 for specification definitions 20-7 PippinHT Operations Manual 460005 Rev G...

  • Page 77: Agarose, High-Pass >30-40 Kb, Marker 75F

    >30000-40000 bp Minimum Size Distribution as CV 50 – 80% Sample Recovery Start Value Accuracy <10% Start Value Reproducibility < 10% Specifications subject to change without notice * see page 20-1 for specification definitions 20-8 PippinHT Operations Manual 460005 Rev G...
  • Page 78 ● ● 500 Cummings Center Suite 2400 Beverly, MA 01915 20-9 PippinHT Operations Manual 460005 Rev G...

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