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Updated 7/7/11

DNA Size Selection System

Operations Manual

Software v. 3.92

Updated 7/7/11

Copyright 2011 Sage Science and Pippin Prep are trademarks of Sage Science Inc., Suite 3150, 500 Cummings Center, Beverly, MA. All rights reserved.

Updated 7/7/11

TABLE OF CONTENTS

INTRODUCTION ................................................................................ 1-1 1

SAFETY AND PRECAUTIONS ......................................................... 2-1 2

UNPACKING AND INSTALLATION .................................................. 3-1 3

ACHIEVING BEST RESULTS FROM THE PIPPIN PREP ................. 4-1 4

PREPARING AND LOADING A CASSETTE .................................... 5-1 5

SAMPLE PREPARATION ................................................................. 6-1 6

SAMPLE COLLECTION .................................................................... 7-1 7

SYSTEM VALIDATION ...................................................................... 8-1 8

WRITING AND EDITING A PROTOCOL ........................................... 9-1 9

RUNNING A PROTOCOL ................................................................ 10-1 10

REVIEWING RUN LOGS ................................................................. 11-1 11

SOFTWARE OVERVIEW ................................................................ 12-1 12

RUNNING IN MANUAL MODE ........................................................ 13-1 13

MANAGING RUN FILES ................................................................. 14-1 14

UPGRADING SOFTWARE .............................................................. 15-1 15

WARRANTY AND SERVICE ........................................................... 16-1 16

ORDERING INFORMATION ............................................................ 17-1 17

INSTRUMENT SPECIFICATIONS ................................................... 18-1 18

SPECIFICATIONS FOR 3% GEL .................................................... 19-1 19

SPECIFICATIONS FOR 2% GEL CASSETTES .............................. 20-1 20

SPECIFICATIONS FOR 2%EF (ETHIDIUM-FREE) CASSETTES ... 21-1 21

SPECIFICATIONS FOR 1.5% GEL CASSETTES ........................... 22-1 22

SPECIFICATIONS FOR 0.75% GEL CASSETTES ......................... 23-1 23

1-1

Introduction 1

Thank you for purchasing the Pippin Prep from Sage Science. The Pippin Prep is the first system to automatically fractionate and purify DNA according to electrophoretic mobility in an agarose gel matrix. We urge you to read this manual to familiarize yourself with the system’s capabilities, precautions, and support.

System Overview The Pippin Prep system features a proprietary five-lane pre-cast agarose gel cassette, and an instrument that combines an electrophoresis power supply, a fluorescence-based DNA detector, and single-board PC. Up to four samples may be processed per run – the fifth lane is used for molecular weight markers (supplied with the cassettes). Each lane is physically isolated from the others, thereby preventing sample cross contamination between lanes. The cassette contains the fluorescent DNA stain, ethidium bromide, which renders the sample DNA and markers detectable by fluorescence optics in the Pippin Prep instrument. During the run, fluorescence data from each lane are recorded by the instrument and analyzed by an on-board computer. Pippin Prep system software uses these data to estimate DNA size of samples in each lane, in real time. The on-board computer also controls the electrophoretic movement of the DNA samples into a buffer-filled elution chamber according to user-programmed values. At the end of the run, extracted DNA is recovered from the elution chambers using standard pipettes. The extracted DNA is recovered in an electrophoresis buffer that is compatible with standard enzymatic treatments such as PCR amplification and ligation. There are three components to the Pippin Prep system:

Instrument – The instrument contains an epifluorescence detector, an electrophoresis power supply, an electrode array, and a Linux-based single-board computer. The system computer is accessed by an external LCD monitor, mouse, and keyboard. The electrode array is located in the top of the sliding instrument cover and is not user-accessible.

Gel Cassette – 5-lane disposable cassettes are manufactured and sold by Sage Science. Each cassette contains precast gel and electrophoresis buffer. External size standards and sample loading buffer are supplied with each cassette kit. Cassettes are available in several gel concentrations and types to accommodate different applications.

Software – System software allows the user to enter the desired DNA size

range for collection, and automates the entire preparative electrophoresis process. The software also logs fluorescence and current data for post-run analyses and QC.

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Safety and Precautions 2

Icons Used In this manual, the following icons will be used to provide the user with information pertinent to the use of the Pippin Prep.

Caution! Warns the user that injury or instrument damage may occur if the contents of the warning are not properly followed.

High Voltage! Warns of the risk of electrical shock if the contents of the warning are not properly followed.

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 system or applications for which is used.

Safe Use Guidelines The Pippin Prep is designed to operate under the following environmental conditions:

Indoor use

Ambient temperature 10-32oC

Humidity 10-80%, non condensing Standard laboratory precautions should be taken when handling Pippin Prep Gel cassettes and operating the Pippin Prep:

Wear a lab coat, safety glasses, and gloves.

Use in proximity of an eye wash station and/or running water.

Important! Running buffer should be disposed of according to internal lab safety policy. Running buffer in some cassettes contain up to 5 ug/ml of ethidium bromide. Cassettes should be disposed of as laboratory waste. All governmental or institutional regulations regarding disposal should be followed.

3-1

Unpacking and Installation 3

Unpacking the Pippin Prep The Pippin Prep Instrument The Pippin Prep instrumentation is shipped in two boxes: one will contain the Pippin Prep and Accessories and the second 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 monitor

Video cable

Power cord

Pippin Prep

Pippin Prep Instrument

Accessory box o Computer keyboard, USB o Computer mouse, USB o Power supply o Power cord o Rinse cassette

3-2

Setting up the Pippin Prep 1. Remove the accessory box located on inside edge of box at the front end of the

instrument. Remove the keyboard, mouse, and instrument power supply from their packaging.

2. Firmly grip both sides of the instrument and lift it from the foam packaging insert. The Pippin Prep weighs approximately 14 lbs. Place the Pippin on a table or bench top.

3. Remove the computer monitor from the manufacturer’s box and connect the monitor to Pippin Prep using supplied video cable. Connect monitor to power using power supply supplied with monitor.

4. Insert USB connector from computer key board into port located in the back of the Instrument.

5. Connect mouse to Pippin Prep via USB or PS2 connector at back of instrument.

6. Connect monitor cable into the video port located in the back or the instrument.

7. Connect monitor to power using power supply and cords supplied with monitor.

8. Connect Pippin Prep instrument to power via supplied power supply and cable. Power connector is at rear of instrument.

9. Press power switch located on the rear of the instrument, and wait for software to launch (approximately 30 seconds).

The Pippin Prep is ready for use. The software should automatically launch – allow 30 seconds.

Figures 1 and 2 on the next page show the rear panel of the Pippin Prep. Figure 3 on the following page shows the front panel.

3-3

Power Entry Port Power Switch Figure 1. Rear Panel Ports

Figure 2. Rear Panel Connections

USB Ports (4) VGA monitor port

Power Cable

Monitor Cable Key board

Mouse

3-4

Figure 3. Pippin Prep front panel.

USB Port

Indicator Light: Blue when instrument is on.

Indicator Light: Green when instrument is running.

3-5

Unpacking Gel Cassettes Gel cassettes are shipped in boxes in one of the following configurations. Ensure boxes are in the upright position and confirm that following contents are present. DNA standards should be stored at 4oC. The cassettes and the remaining reagents stored at room temperature:

10 cassettes with reagents to run 40 samples

o 10 foil-sealed gel cassettes (store at R.T.) o 1 reagent kit for 10 cassettes (store at 4 oC)

200μl DNA size markers 200μl loading solution 50 ml of running buffer

Important! DNA size ladders should be refrigerated at 4

oC.

Important! Cassette should be stored at room temperature with the foil packaging. They are light sensitive and should not be removed until prior to use. Shelf life is six months

4-1

Achieving Best Results from the 4

Pippin Prep

The Pippin Prep extracts narrow or wide DNA size distributions more reproducibly and with higher yield than manual preparative gel techniques. However, due to the unique design of the pre-cast gel cassette, there are key differences between running the Pippin and other agarose gel methods.

It is recommended that users closely follow the instructions outlined in the following section (Section 5) until they become thoroughly familiar with the system. The Quick Guide (included with cassettes) provides protocol reminders, but is not intended for beginning users. The keys to successful operation are:

Preparation of the cassettes (See Section 5)

Sample loading (See Section 5)

Purity of the input sample (See Section 6)

Common Misconceptions Users should be aware of the following characteristics of the Pippin Prep System. These are part of normal operation of the system, but may seem counterintuitive at first.

Narrowest is not always the best. The Pippin Prep can produce very narrow size distributions from sheared genomic DNA. However, narrower size distributions will necessarily mean that a smaller fraction of 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.

DNA undergoing elution is smaller than DNA at the detector. The branch point between the separation and elution channels is downstream from the detector position. Figure 4 shows a cassette channel to illustrate. During normal operation, the leading edge of the DNA fraction scheduled for elution passes the detector before the start of elution (by up to several minutes). This offset can give rise to the impression that sample elution is late, even in runs that are functioning properly. The “BP at Branch” indicator on the home screen shows the size of DNA passing the branch point of the marker lane, and can be used as a real-time check on elution timing.

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Figure 4. An illustration and software display of the time and base pair difference between the detector and display.

Elution creates a jump in the optical background of the sample lanes. Since ethidium is positively charged, it will migrate toward the (-) electrode during electrophoresis – moving in the opposite direction from the DNA. During the initial phase of a Pippin Prep run, the separation channel will become partially depleted of ethidium. However, when elution begins, a front of fresh ethidium from the unused elution channel will migrate up through the separation, past the detector, and create a jump in the ethidium background signal, which can be misinterpreted as a DNA signal. This is illustrated in Figure 5 (next page). The ethidium front passes the detector approximately 4-5 minutes after start of elution. The ethidium front may not be noticeable when DNA input is high.

4-3

Figure 5. Relatively low input sheared DNA sample with broad size

distributions (30-500 bp) in lanes 2-5. Sample loading is the same in all lanes but different DNA ranges were eluted. Brighter bands are ethidium fronts due to elution.

Ethidium fronts due to elution

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Preparing and Loading a Cassette 5

Visually Inspect the Cassette Caution! Standard cassettes contain ethidium bromide. Use appropriate protective equipment (disposable gloves, lab coat, and lab glasses) when handling cassettes.

1. Remove the cassette from foil packaging. Foil package is scored at one

end to allow tearing. 2. Inspect the levels of buffer in the buffer reservoirs. Tip the cassette to

consolidate any bubbles in the reservoirs, then hold the cassette in a horizontal position and look at the reservoirs from the top and bottom 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 reservoir should be refilled prior to running. When refilling, a visual check of buffer levels is sufficient -- a measured volume is not required.

Figure 6. Low buffer levels in cassette buffer chambers.

3. Inspect the gel columns. Look for obvious breakage of the agarose column

in each channel.

Important! If there is obvious breakage, do not use the lane. Remaining lanes can be used.

buffer volumes are Low

(+) side of cassette

(-) side of cassette

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4. Inspect bottom of cassette for bubbles in the detection region of the gel columns. If a cassette has been jarred during shipping, the agarose column can delaminate from the plastic bottom of the separation channel, forming a thin flat bubble between the gel column and the bottom of the channel. If this happens in the region used by the optical detector (see illustration), DNA detection will be extremely unreliable. If a lane with such a bubble is used for the reference marker, the markers will not be properly identified and sample collection will occur at the wrong time or fail altogether. (Only the optical properties of the channel are affected -- electrophoretic properties of the channel are unaffected, 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 tilting the cassette back and forth (see Figure 7, below). Figure 8 shows the region of optical detection.

Important! If a bubble is observed, do not use the lane to run the reference marker or for peak capture mode applications. It may be used for size-based fractionations (tight or bp range modes). Important! Flat bubbles between the top surface of the gel column and the plastic top of the channel will NOT affect run quality or optical detection. They may be used for markers or sample without any adverse effects.

Figure 7. Bubble in optical path. Do not use the lane for the DNA reference marker.

Figure 8. The region of optical detection

Agarose Delamination Do not use this lane for DNA marker

Optical Region

5-3

Prepare the Cassette for Loading 5. Dislodge bubbles from behind the elution wells. Tilt the cassette,

sample well side down, to release the any trapped bubbles behind the elution modules into the (+) buffer chambers. Gently tap the cassette if necessary. Figure 9, below, shows location to check for bubbles.

Figure 9. Diagram of a Gel Cassette.

6. Place Cassette into the Pippin optical nest. The cassette should be

placed into the nest the orientation shown in Figure 9, above, with the sample wells to the left side of the nest. When inserting the cassette into the nest, keep the (-) buffer chambers tilted down so that the bubbles in the elution reservoirs won’t be trapped behind the elution modules.

7. Remove adhesive strips from cassette. Place one hand on the

cassette, and hold it firmly in the nest. Grab the white tabs of the tape and pull the strips firmly and slowly toward the front of the Pippin Prep until they are removed. If there are individual seals over the elution wells, do not remove them.

Important! Cassettes that have been shipped prior to June, 2011 with have open elution wells when the tape is removed. Beginning sometime in Q’3, 2011, cassettes will be shipped with individual tape seals over each elution well. These seals should be kept in place until the end of the run, and removed just prior to sample retrieval.

Important! If there are individual seals over the elution wells, skip step 4 and continue with step 5 (see warning above)

( - ) ( + )

5-4

8. Remove buffer from elution modules and replace with 40μl of fresh electrophoresis buffer. Replenishing the buffer ensures proper electrophoretic continuity. When refilling empty modules, place tip all the way into the module and slowly fill from the bottom, withdrawing the pipette tip while dispensing, and taking care not to generate any bubbles that can occlude the current path.

Important! Make sure that the pipette tips used for step 8 extend all the way to the 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 completely. Test tip fit using the empty rinse cassette supplied with the instrument.

Information. The total volume of the elution module when filled to the top is

65l. The specified starting volume of 40l only partially fills the module.

9. Check the buffer level in the sample wells. Immediately prior to loading,

sample wells should be completely filled to the top with buffer. If any wells are under filled, top them up with additional buffer.

Information. The total volume of the sample well is approximately 70l.

10. Perform the continuity test. Close the lid and press the “Test” button located in the lower right area of the Main screen. The test routine runs automatically and measures the current in each separation and elution channel. If the measured current values fall within the expected values the test returns a “PASS” message, and the cassette is ready for sample loading. If the cassette does not pass, follow the instructions at the end of this Section to troubleshoot.

Important! Temperature dramatically affects electrical current. Cassettes will fail the current test due to low current if they are below 17oC (62oF). Cassettes should be stored at room temperature. If ambient temperature is below 17oC, increase the room temperature, or move the instrument to warmer laboratory locations.

5-5

Program or select a size selection protocol (see Section 9 for details on creating and editing protocols).

1. Load Samples Proper sample loading is critical for best

performance of Pippin Prep cassettes. For maximum reproducibility and accuracy, the sample should travel through the central section of the gel column, and should be bounded on all sides by uniformly conductive media – either gel or electrophoresis buffer. The goal of the loading procedure is to produce this geometry in the sample loading well, as illustrated in the bottom section of Figure 10.

Properly prepared samples will be 40 l in total volume consisting of

30 l of DNA mixed with 10 l of Pippin Prep loading solution. The loading solution contains concentrated Ficoll as a densifying agent (see following Section on Sample Preparation for details), 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 conductive buffer over the sample, the electrophoretic forces lines will curve upward as the sample exits the well (see Figure 10, upper section), and the sample will be drawn to the top of the cassette where it can travel out of the gel into the gap between the gel column and the plastic top of the channel. Sample moving in this gap will travel at a different rate than the sample inside the gel column, and will lead to elution of undesired size fractions in the eluted material. In such cases, the contaminating DNA will usually (but not always) be higher in molecular weight than the selected DNA.

Figure 10. An illustration of the electrophoretic effect on a DNA sample when a sample well in not completely filled.

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11. Re-check the buffer level in the sample wells. Make sure that sample

wells are completely full to the top with electrophoresis buffer. Top up with

additional buffer, if necessary. The total volume of the sample well is 70 l.

12. Remove 40μl of buffer from the first sample well, and load 40μl of

sample (or marker) into that well. Take care not pierce the agarose with the pipette tip. There is gel on all sides and bottom of the sample well. In addition, there is an agarose “chimney” surrounding the top of the sample well that protrudes up through the cassette cover (see Figure 10). When removing buffer, some users find it useful to immerse the pipette tip just below the surface of the buffer and follow the liquid level down with the tip as the buffer is removed. When buffer removal is completed, there will be ~30ul of buffer left in the well. When adding sample, place tip of pipette just below the surface of the buffer, and 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.

13. Repeat step 2 for remaining wells. When all wells are loaded, proceed

to start the desired protocol as described in Section 10.

What to do if a Cassette Fails the Continuity Test

The Continuity Test screen (Figure 11, below) displays the test parameters for the electrical current, and the measured currents for each electrophoretic channel (separation and elution) in each lane of the cassette. Depending on which channel has failed, take the following actions. a) If a Separation lane has failed continuity. Do not use that lane. Remaining

passing lanes are suitable for use.

b) If an Elution channel has failed continuity. Completely remove all buffer

from the elution module of the failed lane and refill with 40 l of fresh

electrophoresis buffer. Retest the cassette. If the lane passes, proceed to use the lane as normal. If the lane fails again, do not use the lane for samples. However, the failed lane may be used as the reference lane, since the marker is never eluted.

Figure 11. The Continuity Test screen. The elution channel in lane 1 has failed.

Test parameters

Separation Channel “FAIL” = do not use lane

Elution Channel “FAIL” = use lane for reference or replace buffer and retest

6-1

Sample Preparation 6

Input Sample Characteristics When running the Pippin Prep, 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). High salt concentrations can result in slower than expected DNA mobility.

Protein in the sample: DNA-binding proteins such as ligases or polymerases can affect the mobility of fragments during separation. Proteins can also reduce DNA recovery from the elution module by increasing the binding of DNA to the ultrafiltration membrane 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 obviously important to program accurate size selection settings. Pippin Prep cassettes are calibrated using the Agilent Bioanalyzer to evaluate input and product sizes, and so, for best results, input size distributions should be evaluated using the Bioanalyzer. For low concentration samples, the Agilent HS chip is very useful (see Technote 4 on the Sage website).

Preparing DNA Samples for the Pippin Prep

1. Bring DNA sample up to 30μl with TE. 2. Bring loading solution to room temperature. 3. For each sample, combine 30μl of DNA sample with 10μl of loading solution. 4. Mix samples thoroughly (vortex mixer). Briefly centrifuge to collect.

Recommended sample Load Guidelines for 1.5%, 2%, and 3% Gel Cassettes

Maximum Load: 10g sheared genomic DNA*

Approx. 4 g restriction/PCR fragment

Minimum Load: Low single nanograms

Optical Sensitivity: approx. 200ng sheared genomic DNA approx. 10-15ng single bands, restriction fragments, PCR products, or synthetic DNA fragments

6-2

Sample Loads on the 0.75% Gel Cassettes In the 0.75% gel cassettes, the input load has a large effect size selection accuracy. The Pippin Prep calibration constants for the 0.75% gel cassette are optimized for a

5g load. Lower input loads will size select a larger target than programmed, while higher input loads will produce a smaller target than programmed. Users should refer to the 0.75% gel specifications section (Section 21) and to determine how much of an offset should be expected, and some method development will likely be required to achieve desired results.

7-1

Sample Collection7

Figure 12. A schematic of a Gel Cassette.

1. Samples can be removed from elution modules using a standard 100-200µl

pipette.

Important! Make sure that the pipette tips used for collection extend all the way to the bottom of the elution modules without sealing the elution port opening. If the tips seal the port opening, it will be extremely difficult, if not impossible, to completely recover the contents of the elution module. Test tip fit using the empty rinse cassette supplied with the instrument.

2. If input samples were not deproteinized, additional DNA product can be

washed from the elution modules with a nonionic detergent. After the initial

product is removed (and saved), add 40 l of TE+0.1% Tween 20 to the

elution module. After a brief incubation (~1 min.), remove the TE-Tween solution and combine it with the initial product. In some cases (restriction digests of DNA), DNA recovery from the Pippin Prep can be doubled by this procedure.

3. Samples from standard cassettes without sealed elution modules will be recovered in 40-65 ul of electrophoresis buffer, depending on the length of the elution step. Samples from cassettes with sealed elution modules (due Q’3, 2011), will be recovered in a fixed volume of 40 ul.

4. When all samples are collected, remove used cassette from instrument and dispose of it properly.

7-2

Important! Eluted samples should not be left in the cassette for longer than a couple of hours to avoid poor recovery (due to adsorption or diffusion). Important! Used cassettes should never be allowed to sit in closed instruments for long periods of time (e.g., overnight). Under those conditions, humidity from the cassette reservoirs can accelerate corrosion of the electrode assembly, located in the sliding cover.

Information. Electrophoresis buffer is 50 mM Tris, 30 mM TAPS, 0.1 mM EDTA. In standard cassettes with ethidium bromide, the concentration is 5

g/ml. During elution, EDTA does not rapidly equilibrate across the ultrafiltration membrane of the elution module, and so the final concentration of EDTA in eluted samples is elevated to 1-2 mM

Information: Eluted samples can be used directly for ligation and amplification without buffer exchange.

Intrinsic Sample Recovery on the Pippin Prep

Intrinsic DNA recovery in Pippin Prep cassettes is determined by running known amounts of a plasmid restriction digest on the Pippin Prep, collecting a broad range of fragments from the digest, and comparing the input and product profile quantitatively using the Agilent Bioanalyzer 2100. The intrinsic recovery of DNA fragments on all Pippin cassette types is between 50-80%. Figure 13, below, shows the recovery of a 5kb band from a 0.75% cassette.

Figure 13. An example of the intrinsic recovery of 5 kb band on a 0.75% agarose cassette.

7-3

Improving Product Yield by Selecting a Wider Size Range The Pippin Prep can produce very narrow size distributions from sheared genomic DNA. However, narrower size distributions will necessarily mean that a smaller fraction of 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 16 shows an example of the effect of size range selection vs. yield.

Figure 16. Increasing yield by widening the size selection range.

8-1

System Validation 8

Control DNA can be purchased from Sage Science to check the performance of the Pippin Prep system (See Section 15, Ordering Information, for the part numbers for these kits). The Control DNA is useful to test, refine, and troubleshoot Pippin Prep size fractionation protocols. For comparison to factory tested cassettes, an extraction protocol is provided, as is typical Agilent Bioanalyzer results. Examples are illustrated below. Pippin Prep cassettes and instruments are functionally tested using restriction digests of genomic 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, without any significant peaks or discontinuities. Following restriction digestion, the control DNA is purified by phenol:chloroform extraction, dialyzed, and diluted into Pippin Prep electrophoresis buffer (without ethidium bromide). The DNA is premixed with Pippin Prep loading solution and is provided ready for loading – no additional loading solution should be added. The DNA concentration is 5 micrograms per 40 microliters. 40 microliters of control DNA should be used per lane. Each tube contains sufficient volume for 16 sample lanes. Control DNA profile (with DNA Marker B) for 2.0% Agarose Cassettes

8-2

Typical Bioanalyzer Result, 150 bp target, Control DNA extracted from a 2.0% Agarose Cassette

Important! Data are not intended to imply guaranteed results or performance. Control standards are intended to demonstrate that the Pippin Prep system is functioning as expected, and that proper operational technique is being used.

9-1

Writing and Editing a Protocol 9

1. Go to Protocol Editor Screen (press “Protocol” on controller of Main Screen).

2. Select “New” to begin writing a new protocol or “Load” to edit and existing protocol.

3. Select a Cassette Type from “Cassette Type” drop down menu.

4. Select a Reference Lane. The DNA Marker provided with cassettes will be loaded on this lane. Press the button on the Ref Lane field. Text may be entered into the “Sample ID” field and saved to the protocol.

5. Programming a Minimum (Tight) Cut. If the peak size of a fragment is known, or if a minimum size range is targeted from genomic DNA, enter a value into the BP Target field. When this value is entered, the minimum size range values for peaks or size distributions at that targeted value will be calculated by the Pippin software. The minimum start and end base pair values will auto-fill the BP Start and BP End fields.

9-2

When a Minimum Size Range has been programmed into a sample lane, the “BP Range Flag” field for that sample will display “tight” and a green color.

The “BP Start” and “BP End” range values may be edited. If the range is changed, the “BP Target” value will automatically change to the average value between the new range. Text may be entered into the “Sample ID” field and saved to the protocol.

6. Programming a Broad Range Cut. Larger cut ranges are programmed by entering

values into the “BP Start” and “BP End” fields. The “BP Target” field will auto-fill with the average value of the entered range.

When a larger size cut range (larger than the minimum) has been programmed into a sample lane, the “BP Range Flag” field for that sample will display “broad” and an orange color.

The “BP Target” range may be re-edited directly. If the target is changed, the “BP Start” and “BP End” values will automatically revert to the minimum range values.

9-3

There is a maximum elution time, beyond which the elution chamber will overflow. The maximum elution time depends o the cassette type and the target range for collection. Should the maximum be exceed, the “BP Range Flag” field for that sample will display “overflow” and a pink color. If an “overflow” condition has been entered, the protocol will not save.

Text may be entered into the “Sample ID” field and saved to the protocol.

7. Programming a Pause. A sample elution may be programmed to pause. A programmed pause in any sample will pause the entire instrument at that time point, and continue the protocol when resumed. To enter a pause, a value must be entered in the “BP Pause” field, and a the value must be within the entered that is within the programmed “BP Start” and “BP End” Range. When a pause has been programmed, a yellow indicator light will appear in the “Pause Enabled” Field, and a warning will appear in the Message Box.

Important! A pause will require the user to manually resume the protocol from the Main Screen controller. The instrument lid may be opened during the pause, and sample retrieved.

Note: A pause will allow a user to remove two consecutive cuts. The cuts ranges will likely have a high degree of overlap. User may wish to use the pause feature for two reasons: 1) to retrieve nearly identical cuts from the same sample, or 2) to recover some of the starting sample.

9-4

8. Saving a Protocol. After programming a new protocol, or editing an existing one, the

protocol file must be saved prior to use. If the protocol is new, press “Save”. If a protocol has been edited, a yellow alert will be displayed in the “Process Changed” field. “Save” will save the file under the previously saved name, and “Save As” will allow a new name for the file to be applied.

All protocol files are saved in single directory.

10-1

Running a Protocol 10

1. Go to the Main Screen (press “Return” if in the Protocol Editor Screen).

2. If necessary, select the protocol to be run from the “Protocol Name” drop down menu in the Run File Manager.

3. Press “Start” on the Controller.

Monitoring a Run

The Sample Monitor has four indicator lights per lane. When the instrument is idle, all “Idle” indicator lights are light gray.

When a protocol is running, the following indicator panel will be displayed:

10-2

Light Gray, Reference. If a lane running the reference DNA Marker, the “Reference” indicator will be light gray.

Green, Separation. If a lane is separating fragments, the “Separate” indicator will be green.

Orange, Elution. If a lane is eluting a size range, the “Elute” indicator will be orange.

Note: If there is a value in the “Elution Timer” field, and the “Separate” indicator is green, then the elution has been completed.

The Elution Timer records the length of time of the elution. When elution is complete, electrophoretic path will switch back to the separation channel.

At the end of every run, the Pippin Prep will automatically save a log file and a screen image of the Main Screen after the run has been finished. The files are saved internally on the Pippin hard drive in the directory named /home/pippin/PippinPrep/Logs/ in a folder with a year-month (YYYY-MM) stamp. Log and image files are saved as .txt and .png files, respectively. Log files have a year-month-day-time stamp (yyyy-mm-dd-hh-mm-ss) stamp. Logs may be accessed from the “MANAGE LOGS” button in the Main Screen, or the “Log File” folder icon in the Review Logs Screen.

11-1

Reviewing Run Logs 11

Note: A stand-alone Review Log Program is available for PCs. Go to www.sagescience.com or contact sage science support.

1. From the Main Screen, select “REVIEW” from the controller.

2. This will launch the log review screen.

3. To select a log file, press the folder icon in next to the “Log File” field.

http://www.sagescience.com/

11-2

4. Select a log (.txt) file.

5. This will populate the Review Log Screen with data from the selected run log:

6. In addition to view run data, the following may be accomplished with in the Log Review Screen:

Scale in the y-direction

Graphically manipulate traces

Re-analyze DNA marker peak detection

Copy DNA marker size vs. time to clipboard (for PC only, to paste into worksheets

Optical Traces

Electrophoretic Current Traces (current drops during elution)

Protocol

DNA Marker Times

12-1

Software Overview 12 Overview

The Pippin Prep uses two main software screens; one is used for running and monitoring (), and the second is used for writing and editing protocols (Editor).

Main Screen

Protocol Editor

Graph

Image

Sample Monitor

Run File Manager

Controller

Protocol Name

Size Range Selector

Cassette Selector

Editor File Manager

Message Box

12-2

Main Screen Detail

Graph Displays the signal output of the optical detectors as a graph.

Image Displays the signal output of the optical detectors as an image.

`

12-3

Sample monitor Displays the status of individual samples. Controls individual lanes in Manual Mode.

Sample ID: Displays the sample ID that was entered into protocol. Current: Displays the electrical conductivity within the lane (mA). Fluor: Displays the optical detector value (fluorescence units). Elution Timer: Clock display of the duration of an elution event. Reference: Indicates the lane that has been selected for reference. Idle: Indicates whether a lane is idle or running. Stops electrophoresis in Manual Mode. Separate: Indicates the electrophoretic path is applied to separation channel. Starts separation channel in Manual Mode. Elution: Indicates the electrophoretic path is applied to elution channel. Starts elution channel in Manual Mode.

Run File Manager Selects run protocols and provides options for run logging.

Protocol Name: Selects the protocol to be run.

Progress: Indicates duration of current run, and % progress of overall run time. Peaks Detected: Indicates the number of reference peaks detected in size ladder. Voltage: Displays electrophoresis voltage of run. Clock: Displays current time. Save Graph: Saves graph of previous run as an image file. Save Image: Saves image of previous run as an image file. Save Window: Saves a screenshot of the current screen as an image file. Manage Logs: Opens a screen for transferring (to USB storage) log and image files.

12-4

Controller Controls operation of the Pippin Prep instrument.

Test: Tests electrophoretic currents in gel cassette Start: Starts a run.

Pause: Pauses (and resumes) a run. Stop: Stops a run. Protocol: Go to Protocol Editor Screen. Review: Go to Log Review Screen

Info: Displays information about the Pippin Prep system, and links to software update screen. Shutdown: Shuts down the Pippin Prep system.

Protocol Editor Screen Detail

Cassette Type Selector Selects cassette type on which protocol will be run.

Size Range Selector User inputs values to select size ranges to be eluted, selects reference lane, and/or programs a pause during an elution event.

Run Time: Adjusts the total run time, defaults to 2 hours.

BP Target: Edits and displays peak size or center fragment length to be eluted. BP Start: Edits and displays the starting value of fragment size range to be eluted. BP End: Edits and displays the ending value of fragment size range to be eluted. BP Pause: Edits and displays the value of fragment size range when elution is paused. Sample ID: Input sample identification. Reference: Selects and indicates lane used for reference (running size ladder). Pause: Indicates that a pause has been programmed into a lane’s run.

12-5

Message Box

Displays errors or exceptions with Size Range Selector input.

Protocol Name

Displays name of loaded or saved protocol.

Protocol File Manager Displays name of protocol being edited.

New: Clears all fields so a new protocol can be programmed. Load: Loads existing protocol into Editor Screen. Delete: Deletes and existing file. Protocol Changed: Indicates that a change has been made to an existing protocol. Save As: Saves protocol on Editor Screen as a new named protocol. Save: Save protocol on Editor Screen as currently name protocol. Return: Returns to Main Screen.

13-1

Running in Manual Mode 13 The Pippin Prep may be run without using a programmed protocol and with manual control. 1. Select the Manual Mode from the “Protocol Name” drop down menu in the Run File

Manager.

2. Sample lanes may be run individually by pressing the “Separate” button and elutions are carried out by pressing the “Elute” button. After elution, the “Separate” button, to continue separations, or “Idle” button will stop the run on that lane.

Timing a cut in Manual Mode. 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 be eluted. It should assumed that a fragment will in position to be collected approx. 6 minutes after it is detected. Elutions should be undertaken for at least 4 minutes or no sample will be collected. After 4 minutes, size ranges will be collected at a constant rate. For 2% gels (check cassette documentation for other formulations), collections ocuur at 8 bp/mi

14-1

Managing Run Files 14

File Types Every run on the Pippin Prep automatically saves two run files in a single directory. Both files will share the same date and time stamp:

Log File. This is a text (.txt) file that contains all of the data that was collected during a run. This file can be used as a diagnostic tool with Sage Science support personnel. Data from the log file can be viewed in the Log Review Screen, or copied into a spreadsheet file to recreate the data displayed in the Pippin Prep graph images.

Screen Image File. An image file (.png) of the Main Screen after each run has been completed is save in the same directory as log files.

In addition, a Graph or Image may be manually saved as individual image (.jpg) after a run is complete. In the Main Screen go to the Run File Manager. Press “Save Graph” to save an image of the Graph, press “Save Image” to save an image of the Image, and press “Save Window” to save an image of the entire Main Screen.

14-2

Transferring Log files to USB device Files are saved in a source directory named “Logs” in a folder that is named by month (YYYY-MM). A new folder is automatically created every month. To transfer files from the Pippin Prep: 1. Insert a USB storage device in the USB port on the front panel of the instrument (there

are additional ports on the rear panel).

2. In the Run File Manager Press “Manage Logs”. This will launch the File Transfer Window.

3. Open a folder by double clicking. Select files to be transferred by ctrl-click or shift-click.

Press “Copy Selected Files”. Selected files will copy to the target USB device. 4. Press “Unmount Flash Drive”. 5. Remove USB device.

The Manage Files Screen also has the following functions.

New Target Folder. Allows users to create and name a new folder on the destination USB storage device.

Delete Selected Files. Permanently deletes files that have been selected.

Return. Returns to the Main Screen.

15-1

Upgrading Software 15

1. Insert USB drive into port on front panel of Pippin Prep (software file must be in root directory).

2. From the main screen, press “INFO”, this will launch the information window.

3. From the Information Window, press “SOFTWARE UPGRADE”

4. Press “OK” at the warning prompt, the Software Upgrade Window will launch.

15-2

5. Press “OK” at the warning prompt, the Software Upgrade Window will launch.

6. Press “INSTALL UPGRADE”, this will take about 5 seconds

7. Press “UNMOUNT FLASH DRIVE”

8. Press “EXIT” to return to Main Menu. This will install the software. Installation may take up to 30 seconds.

9. Check the upper right hand corner of the Main Screen to confirm that the proper software version has installed.

Note: The system should be ready to use re-boot or power cycle is not necessary.

16-1

Warranty and Service 16 Warranty Sage Science will offer a no charge repair or replacement of defective product if notified within the warranty period and product has not been misused, altered or damaged by disaster. This warranty is not transferable from the original purchaser to a subsequent purchaser.

Instruments – The Pippin Prep instrument and monitor have a standard 2-year parts and labor warranty. Instruments are subject to depot repair or replacement. On-site maintenance or repair is not implied. Do not attempt to repair or disassemble the Pippin Prep instrument. This will void the remaining warranty and possible 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 Pippin Prep contains high voltage apparatus. The instrument should be repaired by authorized personnel only. Do not disassemble under any circumstance.

Cassettes – Cassettes have a 6-month warranty policy. If a cassette or sample lane is defective Sage Science will replace the cassette at no cost. The defective cassette should be disposed of as usual. Sage Science support staff may ask for a log file or image file to evaluate or troubleshoot cassette issues.

Maintenance It is possible that salts will accumulate on the electrodes that are housed in the Pippin Prep lid assembly. This may alter the performance of the instrument over time. It is recommended that the electrodes undergo periodic rinsing. To rinse the electrodes, use the rinse cassette provided in the instrument shipment, fill with deionized water, load on the sample tray and close the Pippin Prep lid. A quick rinse for several second is sufficient. If a rinse cassette is lost or damaged contact Sage Science support for a replacement.

No other maintenance activity is required.

Cassette Disposal Running buffer should be disposed of according to internal lab safety policy. Running buffer contains up to 5 ug/ml of ethidium bromide. Cassettes should be disposed of as laboratory waste.

17-1

Ordering Information 17 Pippin Prep Instrument PIP0001 (Includes monitor, keypad, mouse and accessories) Gel Cassettes (Includes loading buffer, DNA size ladder and extra running buffer) 0.75% Gel Cassette 10/pk CSD7510 1.5% Gel Cassette 10/pk CSD1510

2% Gel Cassette 10/pk CSD2010 2% Gel Cassette, Ethidium-free 10/pk CEF2010 3% Gel Cassette 10/pk CSD3010

Control DNA for Performance Validation (e.coli DNA digest for 16 sample loads [5 ug/load]) Control DNA for 0.75% Gel Cassette CON7504 Control DNA for 1. 5% Gel Cassette CON1504 Control DNA for 2.0% Gel Cassette CON2004 Control DNA for 3.0% Gel Cassette CON3004 Reagent Replacement Kits

(Loading buffer, DNA size ladder and extra running buffer for 10 cassettes. Replaces expired or misplaced kits that are included with Gel Cassette orders)

Reagent Replacement Kit for 0.75% Gel Cassettes RPK7510 Reagent Replacement Kit for 1.5% Gel Cassettes RPK1510 Reagent Replacement Kit for 2% Gel Cassettes RPK2010 Reagent Replacement Kit for 2%EF Gel Cassettes REK2010 Reagent Replacement Kit for 3% Gel Cassettes RPK3010

18-1

Instrument Specifications 18

Specifications for Pippin Prep Instrument

Electrophoresis Voltage 100 V, constant Typical current per sample lane 2.0 mA Optical detection 535 nm excitation, 640 nm emission Power Requirements 100-240 VAC, 2.5 A, 50-60 Hz Weight 15 lbs Dimensions 7” H X 11” W X 21” Approvals CE, CSA Country of Origin United States

19-1

Specifications for 3% Gel 19

Cassettes (Check www.sagescience.com for newly available gel formulations)

DNA Size Selection Selectable target size ranges 50 - 300 bp Run times 57 – 135 min.

Minimum size distribution 5.0 % at 50 bp as expressed by (CV) 3.5 % at 300 bp

Maximum collected size range 250 bp Accuracy + 5% Reproducibility + 5%

Sample Recovery 50-80%

Sample Loading

Maximum load capacity 10 g sheared genomic DNA

4 g DNA band

Minimum load capacity low single nanograms

Optical sensitivity Approx. 200 ng sheared genomic DNA Approx. 10-15 ng single band, restriction fragments, PCR products, or synthetic DNA fragments

19-2

Approximate Run Times, 3% Agarose

Target Range: 50 – 300 bp Run Times: 57 – 135 min.

Pippin Prep Run Display DNA Reference Marker, 3% Gel (bp)

Marker C

Specifications subject to change without notice

Size, bp

Time to detector

(minutes)*

Time to collect

min. range (minutes)*

20 45 NA

50 50 57

75 62 70

100 70 79

*run times vary based on temperature and other factors. This is an approximate guideline – actual values may vary up to +5 min.

0

50

100

150

200

250

300

40 60 80 100 120 140

Target (bp)

Minutes

Time to Collection

("tight" cut)

Time to Detector

20-1

Specifications for 2% Gel Cassettes 20





Sample Loading


4 g DNA band



20-2

Approximate Run Times, 2% Agarose


Pippin Prep Run Display

DNA Reference Marker, 2% Gel (bp)

Marker B


0

100

200

300

400

500

600

20 40 60 80 100 120 140

Target (bp)

Minutes

Size, bp

Time to detector

(minutes)*

Time to collect


20 37 NA

75 43 NA

150 51 57

300 66 73

600 100 111

Reference DNA Markers, 2% Gel *run times vary based on temperature and other factors. This is an approximate guideline – actual values may vary up to +5 min.

Time to Detector

Time to Collection ("tight" cut)

21-1

Specifications for 2%EF 21

(Ethidium-free) Cassettes





Sample Loading


4 g DNA band


Optical sensitivity Approx. 200 ng sheared genomic DNA Approx. 10-15 ng single band, restriction fragments, PCR products, or synthetic DNA fragments.

21-2

Approximate Run Times, 2%EF Agarose



DNA Reference Marker, 2%EF Gel (bp)

Marker E


Size, bp

Time to detector

(minutes)*

Time to collect


20 32 NA

75 37 NA

150 44 51

300 58 67

600 86 100

*run times vary based on temperature and other factors. This is an approximate guideline – actual values may vary up to +5 min.

0

100

200

300

400

500

600

20 40 60 80 100 120 140

Target (bp)

Minutes

Time to Detector

Time to Collection

("tight" cut)

22-1

Specifications for 1.5% Gel Cassettes 22

DNA Size Selection Selectable target size ranges 300 - 1500 bp Run times 48 – 140 minutes


Maximum collected size range 1000 bp Accuracy + 7% Reproducibility + 7% Sample Recovery 50-80%

Sample Loading


4 g DNA band

Minimum load capacity low single nanograms sheared gDNA 50 ng DNA band


22-2

Approximate Run Times, 1.5% Agarose



DNA Reference Marker, 1.5% Gel (bp)

Marker A


0

300

600

900

1200

1500

20 40 60 80 100 120 140

Target (bp)

Minutes

Size, bp

Time to detector

(minutes)*

Time to collect


75 37 NA

150 41 NA

300 49 53

600 65 64

1200 96 105

1500 110 122**

* run times vary based on temperature and other factors. This is an approximate guideline – actual values may vary up to +5 min.

Time to Detector

Time to Collection

("tight" cut)

** run time setting in software should in be set to 2:30 for large cuts.

23-1

Specifications for 0.75% Gel Cassettes 23

DNA Size Selection Selectable target size ranges 2 - 8 kb Run times 55 – 100 minutes

Minimum size distribution 20 % at 2 kb as expressed by (CV) 15 % 3-8 kb

Maximum collected size range 6 kb Accuracy Accuracy is dependent on input load (see

the chart on page 21-3).

Reproducibility* Sample Recovery 50-80%

Sample Loading


4 g DNA band



Important! Input sample loading of 0.75% gel cassettes effects accuracy In the 0.75% gel cassettes, the input load has a large effect on size selection accuracy. Lower input loads will size select a larger target than programmed, while higher input loads will produce a smaller target than programmed. Users should refer to the chart on page 21-3 to determine how much of an offset should be expected, and some method development will likely be required to achieve desired results. Input amounts that are

below 1g have not fully characterized and may require method development by users.

* Reproducibility is defined as CV of averages from 4 replicate tight collections on the same sample.

bptarget (%)

2000 8

4000 5

6000 5

8000 3

23-2

Approximate Run Times, 0.75% Agarose

Target Range: 2 – 8 kb Run Times: 60 – 100 min.


DNA Reference Marker, 0.75% Gel (bp)

Marker D

Time to Detector (mins) Time at end of elution (mins)

bptarget mean stdev mean stdev

2000 48 2 62 2

4000 60 3 75 3

6000 70 3 87 3

8000 78 4 93 4

Run times on 0.75% gel cassettes


23-3

To use this chart: Find the desired target size on the y-axis, and follow the value to the approximate input amount. Enter the corresponding x-axis value into the protocol editor software. Each point is an

average of four experiments. Error bars for the 5 g load represents two standard deviations.

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