Bio-Rad Experion DNA Analysis Kits User Manual
Experion™
DNA 1K and DNA 12K
Analysis Kits
Instruction Manual
Catalog #700-7107
#700-7108
Bio-Rad Technical Support
For help and advice regarding products from the Experion™ automated electrophoresis system, please contact the Bio-Rad
Technical Support department, which in the United States is open Monday–Friday, 5:00 AM–5:00 PM, Pacific Time.
Phone: 1-800-4BIORAD (1-800-424-6723)
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Although prepared to ensure accuracy, Bio-Rad assumes no liability for errors, or for any damages resulting from the application
or use of this information.
Excel is a trademark of Microsoft Corporation.
The dyes in Experion kits are manufactured by Molecular Probes, Inc. and are licensed for research use only.
LabChip and the LabChip logo are trademarks of Caliper Life Sciences, Inc. Bio-Rad Laboratories, Inc. is
licensed by Caliper Life Sciences, Inc. to sell products using the LabChip technology for research use only.
These products are licensed under U.S. patents 5,863,753; 5,658,751; 5,436,134; and 5,582,977, and pending
patent applications, and related foreign patents, for internal research and development use only in detecting,
quantitating, and sizing macromolecules, in combination with microfluidics, where internal research and development use
expressly excludes the use of this product for providing medical, diagnostic, or any other testing, analysis, or screening services,
or providing clinical information or clinical analysis, in any event in return for compensation by an unrelated party.
Copyright © 2010 Bio-Rad Laboratories, Inc.
Contents
Chapter 1: Experion™ DNA Analysis Kits.............................................. 1
1.1 Product Description .......................................................... 2
1.2 Kit Components ............................................................. 3
1.3 Storage Conditions ........................................................... 3
1.4 Additional Requirements ....................................................... 4
Chapter 2: Essential Practices ...................................................... 5
2.1 Storing and Preparing Samples and Reagents ...................................... 6
2.2 Priming and Loading the Chip................................................... 6
2.3 Running the Analysis.......................................................... 7
2.4 General Maintenance ......................................................... 7
2.5 Experion Video Tutorials ....................................................... 7
Chapter 3: Experion DNA Assay Procedure ........................................... 9
3.1 Set Up the Electrophoresis Station .............................................. 10
3.2 Equilibrate the Kit Reagents ................................................... 10
3.3 Prepare the Gel-Stain Solution ................................................. 10
3.3.1 Experion DNA 1K Analysis Kit ............................................... 10
3.3.2 Experion DNA 12K Analysis Kit .............................................. 10
3.4 Prepare the Samples and the DNA Ladder ........................................ 11
3.5 Prime the Chip ............................................................. 11
3.6 Load the Chip .............................................................. 12
3.7 Run the DNA Analysis ........................................................ 13
3.8 Clean the Electrodes......................................................... 15
3.9 Evaluate the Run............................................................ 15
Chapter 4: Data Analysis ......................................................... 17
4.1 Viewing Data............................................................... 18
4.1.1 Managing Run Files and Project Folders in the Tree View.......................... 18
4.1.2 General Display Controls .................................................. 19
4.1.3 Electropherogram View.................................................... 19
4.1.4 Gel View ............................................................... 23
4.1.5 Results and Settings ..................................................... 23
4.2 Changing the Fluorescence Intensity Scale ........................................ 24
4.3 Using Results and Settings to View and Annotate Data .............................. 25
4.4 Comparing Data from Different Runs ............................................ 26
4.5 Saving, Exporting, and Printing Data............................................. 27
4.5.1 Saving Data Files ........................................................ 27
4.5.2 Exporting Data Files to Other Applications ..................................... 27
4.5.3 Printing Data Files ........................................................ 27
Chapter 5: Quantitation Methods................................................... 29
5.1 DNA Quantitation Methods .................................................... 30
5.2 Performing Percentage Determination............................................ 30
5.3 Performing Concentration Determination.......................................... 31
Chapter 6: Changing Analysis Settings and Parameters ................................ 33
6.1 Designating and Searching for Specific DNA Fragments .............................. 34
6.2 Manually Setting a Marker..................................................... 34
6.3 Excluding a Peak from Analysis ................................................ 34
6.4 Changing Peak Finding Parameters ............................................. 35
6.5 Changing General Settings .................................................... 35
6.6 Baseline Modification ........................................................ 36
6.7 Turning Analysis Off ......................................................... 37
6.8 Manual Peak Integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Chapter 7: Troubleshooting ....................................................... 39
7.1 Electrophoresis and Priming Stations ............................................ 40
7.2 Experion DNA Analysis ........................................................ 40
7.3 Contacting Technical Support .................................................. 43
Appendices .................................................................... 45
Appendix A: How the Experion System Works ........................................ 46
Appendix B: Deep Cleaning Procedure .............................................. 50
Appendix C: Glossary ........................................................... 51
Appendix D: Ordering Information .................................................. 53
1
Experion™ DNA
Analysis Kits
1
Experion Automated Electrophoresis System
1.1 Product Description
The Experion DNA 1K and 12K analysis kits are used for DNA analysis with the Experion automated
electrophoresis system (Figure 1.1). The Experion system employs LabChip microfluidic technology to
automate electrophoresis and analysis by integrating separation, detection, and data analysis within a
single platform. Using much smaller sample and reagent quantities than standard analysis methods, the
Experion automated electrophoresis system can be used both upstream and downstream of a number
of nucleic acid and protein applications.
The Experion DNA 1K and DNA 12K analysis kits allow analysis of DNA fragments of 15–1,500 bp and
50–17,000 bp, respectively. These DNA assays provide high sensitivity and excellent resolution (down
to 5 bp) over a broad dynamic range (Table 1.1). Consuming only 1 µl of sample for each analysis, the
Experion automated system can analyze 1–11 samples in ~40 min. These assays are recommended
for rapid, efficient analysis of restriction digests, amplified DNA, microsatellites, and amplified fragment
length polymorphisms (AFLPs).
For details about how the Experion DNA analysis kits analyze DNA, refer to Appendix A in this manual.
Register your Experion system to ensure you receive important updates on software, tech notes,
and manuals. Upon installation, a dialog provides registration instructions.
4
1
2
5a
Fig. 1.1. The Experion system. The system includes the following components: 1) automated electrophoresis
station, 2) priming station, 3) vor tex station used for nucleic acid analysis only, 4) system operation and data analysis
tools (software), and 5) analysis kits, which include the (a) chips and (b) reagents for protein (Pro260 kit), standardsensitivity RNA (StdSens kit), high-sensitivity RNA (HighSens kit), and DNA (DNA 1K and 12K kits) analyses.
3
5b
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Experion DNA 1K and DNA 12K Analysis Kits
Table 1.1. Experion DNA analysis kit specifications.
Experion DNA 1K Experion DNA 12K
Number of samples 11 11
Sample volume 1 µl 1 µl
Total run and analysis time 40 min 30 min
Sizing range 15–1,500 bp 50–17,000 bp
Resolution 25–100 bp: 5 bp 100–1,500 bp: 10%
100–700 bp: 5% 1,500–12,000 bp: 20%
700–1,000: 10%
Quantitation range (≥100 bp) 0.5–50 ng/µl 0.5–50 ng/µl
Limit of detection 0.1 ng/µl 0.1 ng/µl
Sizing accuracy ±10% ±15%
Sizing reproducibility CV ≤5% CV ≤5%
Quantitation accuracy (in TE) ±25% ±20%
Quantitation reproducibility1 (in TE) CV ≤20% CV ≤20%
Maximum salt concentration:
KCl or NaCl 200 mM 250 mM
MgCl
1
Applies to inter- and intrachip variation.
2
15 mM 15 mM
1.2 Kit Components
Table 1.2. Components of the Experion DNA analysis kits.
Item Description Volume (per Vial) 10-Chip Kit
DNA chip Microfluidic chips used for DNA separation 10 chips
Cleaning chip Chip used for cleaning electrodes 1 chip
DNA gel Proprietary polymeric sieving matrix 250 µl (DNA 1K) 3 vials
650 µl (DNA 12K) 1 vial
DNA stain Proprietary fluorescent dye 40 µl 1 vial
DNA loading buffer Buffer for sample preparation; contains lower and upper 750 µl 1 vial
markers for alignment of samples to the DNA ladder
DNA ladder DNA standard containing 11 DNA fragments of 25–1,000 bp 20 µl 1 vial
(DNA 1K) or 100–10,380 bp (DNA 12K)
Spin filters Used for filtering reagents during sample preparation 3 filters
1.3 Storage Conditions
Table 1.3. Storage conditions.
Item Storage Shelf Life
Experion reagents 4ºC See expiration date on packaging
Experion chips Ambient See expiration date on packaging
Gel-stain solution (GS, prepared) 4ºC 1 month from filtration; do not refilter
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Experion Automated Electrophoresis System
1.4 Additional Requirements
Experion automated electrophoresis station
Experion priming station
Experion vortex station
Microcentrifuge (1,000–10,000 x g)
Benchtop vortexer
Aluminum foil
Calibrated pipets and DNase-free narrow-bore tips (for example, VWR #87001-688 or Rainin #L-10F)
DNase-free microcentrifuge tubes, 0.65 ml
DNase-free water (for example, Experion DEPC-treated water, catalog #700-7253)
Extra spin filter for additional filtration steps (as needed, catalog #700-7254)
Experion electrode cleaner (catalog #700-7252)
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2
Essential Practices
5
Experion Automated Electrophoresis System
2.1 Storing and Preparing Samples and Reagents
Store all Experion™ reagents at 4°C when not in use. Do not store reagents at room temperature for
>2 hr, as this will shorten their shelf life.
Before use, allow all kit reagents to equilibrate to room temperature (~15–20 min). Once thawed, gently
vortex all kit reagents before use. Before opening the tubes, quickly centrifuge them to collect solution
to the bottoms of tubes.
If the DNA gel has frozen, discard it.
Protect the DNA stain and gel-stain solution (GS) from light: store these solutions in a dark place and
keep them covered with foil when using them.
The DNA stain contains DMSO, which is hygroscopic. Cap tightly.
Use prepared GS for up to 1 month. Discard after 1 month; do not refilter.
Do not use coated or treated pipet tips or microcentrifuge tubes (for example, siliconized polypropylene)
for preparation of kit reagents or samples. Use of treated tips or tubes may cause separation artifacts.
Use DNase-free microcentrifuge tubes, pipet tips, and TE buffer as needed. Do not use autoclaved
water for sample or reagent preparation.
2.2 Priming and Loading the Chip
To avoid contamination, wear gloves and handle chips by the edges. Never touch the glass portions of
the chip.
Load the chip on a benchtop or in the priming station. Never load a chip in the electrophoresis station.
Avoid sources of dust and other contaminants when preparing samples and loading the chip. Foreign
particles in reagents, samples, or the wells of the chip interfere with separation. Remove chips from their
packaging immediately before use.
It may be easier to load the chip on a white background. Tilt the chip to look for bubbles.
Use narrow-bore pipet tips for loading the chip (for example, VWR #87001-688 or Rainin #L-10F).
To avoid introducing air bubbles, do the following (for more help with chip loading, refer to the Experion
Training Video in the Experion software Help section under Contents and Index > Contents >
Appendices > Technical Videos):
n
Insert the pipet tip all the way to the bottom of the chip well when dispensing liquids
(this reduces the possibility of trapping air)
n
Hold the tip vertically, perpendicular to the chip surface. Holding the tip at an angle
may trap air bubbles at the bottom of the well
n
When expelling liquid, dispense slowly and only to the first stop on the pipet. Using the
second stop introduces air and bubbles into the liquid. Reverse pipetting is acceptable
Dislodge bubbles at the bottom of a well with a clean pipet tip, or remove the solution and load it again.
Use a primed and loaded chip within 5 min of loading. When chips are not used within this time,
reagents may evaporate, leading to poor results or a chip performance error.
Fill all the chip wells when running an analysis. Use blank samples (prepared with water instead of
sample) or replicates if necessary. All 16 electrode pins must be in contact with liquid; otherwise, an
IV (current voltage) check failure error will occur.
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Experion DNA 1K and DNA 12K Analysis Kits
2.3 Running the Analysis
Place the electrophoresis station on a stable surface, where it will not be subjected to vibrations or other
movement, and away from direct sunlight and all other potential sources of extreme heat.
Power on the electrophoresis station before launching Experion software.
The first time that the Experion electrophoresis station is used, confirm that communication has been
established between the software and electrophoresis station before preparing the reagents.
Do not open the lid of the electrophoresis station during a run. The run will abort if the lid is opened.
2.4 General Maintenance
For recommendations on general instrument maintenance, refer to the Experion system manual
(bulletin 10001312).
Clean the electrodes after each run (routine cleaning). Cleaning maintains the instrument in optimum
condition and prevents buildup and cross-contamination of reagents and samples.
Perform the deep cleaning procedure described in Appendix B to clean the electrodes:
n
Prior to first use of the Experion electrophoresis station
n
Whenever contamination is suspected or visible (for example, salt deposits or other
precipitates) on the electrodes
n
Whenever a chip has been left in the electrophoresis station for an extended period of time
(for example, overnight)
Never store the cleaning chip inside the electrophoresis station. Store the empty cleaning chip covered
to keep the wells clean. A new cleaning chip is included with every box of chips.
2.5 Experion Video Tutorials
For additional information, view the video tutorials available online at www.bio-rad.com:
North America: Home > Life Science Research > Support > Tutorials > Electrophoresis and Blotting >
Experion System Training
Other: Home > Life Science Research > Electrophoresis > Automated Electrophoresis >
Experion Training Videos
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Experion Automated Electrophoresis System
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Experion™ DNA Assay
3
Procedure
For an abbreviated version of this protocol, refer to the Quick Guide provided with the kit.
9
Experion Automated Electrophoresis System
3.1 Set Up the Electrophoresis Station
1. If needed, perform a deep cleaning of the electrodes (see Appendix B for instructions).
2. Power on the computer and then power on the Experion electrophoresis station by pushing the
green button in the center of the front panel. The steady green LED above the button indicates the
unit is on.
3. Launch Experion software. If the instrument and computer are communicating properly:
n
A green dot and the last 4 digits of the instrument serial number appear at the lower right of the
software screen
n
The electrophoresis station icon appears in the upper left corner
When there is no connection, these indicators are absent and a grayed-out instrument icon appears
at the upper left of the software screen.
3.2 Equilibrate the Kit Reagents
1. Equilibrate the following kit reagents to room temperature for 15–20 min:
n
DNA stain (blue cap)
n
DNA loading buffer (yellow cap)
n
DNA gel (green cap)
2. Invert each tube several times and then vortex to reincorporate any condensed liquid. Briefly
centrifuge the solutions to the bottom of the tubes. Make sure the DNA stain solution (blue cap) is
thawed before proceeding.
3.3 Prepare the Gel-Stain Solution
A gel-stain solution (GS) preparation is sufficient for use with at least four DNA chips. Use the GS
within 1 month. Keep prepared GS at room temperature and covered with foil until ready for use.
If the GS was already prepared, equilibrate it as detailed above.
3.3.1 Experion DNA 1K Analysis Kit
1. Prepare the GS by adding 12.5 µl DNA stain (blue cap) to a tube of 250 µl DNA 1K gel (green cap).
Vortex the GS for 10 sec, and then spin it down briefly in a microcentrifuge.
2. Transfer the GS to a spin filter, and label and date the tube.
3. Centrifuge the spin filter for 15 min at 2,400 × g. Inspect the tubes to ensure all of the gel has passed
through the filters, and then discard the filters. Blue staining of the filter membrane is normal.
4. Wrap the tube of GS in aluminum foil to protect the stain from light.
3.3.2 Experion DNA 12K Analysis Kit
1. Prepare the GS by combining 10 µl DNA stain (blue cap) and 200 µl DNA 12K gel (green cap) in a
DNase-free 0.65 ml microcentrifuge tube. Vortex the GS for 10 sec and then centrifuge briefly.
2. Transfer the GS to a spin filter, and label and date the tube.
3. Centrifuge the spin filter for 10 min at 1,500 × g. Inspect the tubes to ensure all of the gel has passed
through the filters and then discard the filters. Blue staining of the filter membrane is normal.
4. Wrap the tube of GS in aluminum foil to protect the stain from light.
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Experion DNA 1K and DNA 12K Analysis Kits
3.4 Prepare the Samples and the DNA Ladder
Each chip can analyze up to 11 DNA samples. All wells of a chip must be filled for the
electrophoresis station to operate properly.
1. Remove the DNA ladder from storage, vortex to mix, then centrifuge briefly. Place the ladder on ice.
2. Prepare the samples as necessary to meet the following requirements for analysis:
n
DNA concentration — Samples should have a DNA concentration within the linear range
of the assay (0.1–50 ng/μl). Make an appropriate dilution of the sample, for example in
TE buffer (10 mM Tris, 1 mM EDTA, pH 8), as needed
n
Salt concentration — The concentration of salt in the DNA sample must be <250 mM KCl
or NaCl, 15 mM MgCl2. High salt concentrations increase conductivity, reducing the
amount of sample injected onto the chip and affecting assay sensitivity and potentially
reducing quantitation accuracy and precision. Make an appropriate dilution of the sample
in TE buffer or water, as needed
n
Restriction enzyme deactivation — Restriction enzyme digests may require the addition
of EDTA to chelate metal ions and/or heat inactivation of the enzyme prior to running the
chip. Failure to inactivate the restriction enzyme may result in degradation of the internal
DNA markers, inaccurate sizing, or peak broadening
3.5 Prime the Chip
Start the run within 5 min of priming and loading the chip. For help with chip loading, refer to the
Experion Training Video: Chip Loading, available in the Experion software Help section under
Contents and Index > Contents > Appendices > Technical Videos.
1. Pipet 9 µl GS into the highlighted well labeled GS, gel priming well (Figure 3.1). Insert the pipet tip
vertically and to the bottom of the well when dispensing. Dispense slowly to the first stop on the
pipet, and do not expel air at the end of the pipetting step.
2. On the priming station, set the pressure setting to C and the time setting to 3 or 1, as specified by
the alphanumeric code on the chip (Figure 3.1).
Gel priming well
Priming codes
Fig. 3.1. Experion DNA chip. The locations of the gel priming well
(GS, highlighted) and alphanumeric priming codes are indicated.
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Experion Automated Electrophoresis System
3. Open the Experion priming station and place the chip on the chip platform, matching the arrow on
the chip with the alignment arrow on the chip platform. A post on the chip prevents insertion in the
wrong position. Do not force the chip into position.
4. Close the priming station by pressing down on the lid. The lid should snap closed.
5. Press Start. A “Priming” message appears on the screen of the priming station, and the timer counts
down. Priming requires 30 or 60 sec. Do not open the priming station during countdown.
6. An audible signal and “Ready” message indicate that priming is complete. Open the priming station
and remove the chip. If the lid sticks, press down on it while pressing down on the release lever.
7. Turn the chip over and inspect the microchannels for bubbles or evidence of incomplete priming.
If the chip is primed properly, the microchannels are difficult to see (compare a primed chip to a new,
unused chip). If you detect a problem, such as a bubble or incomplete priming, prime a new chip.
Bubbles forced into microchannels during priming take the shape of the microchannel and are
elongated, not round.
8. Place the chip on a clean surface for loading.
3.6 Load the Chip
1. Pipet 9 µl GS into the 3 other wells labeled GS (Figure 3.2).
2. Pipet 5 µl DNA loading buffer (yellow cap) into each sample well and the ladder well (wells 1–11 and
L, Figure 3.2) .
Use a new pipet tip for each delivery to prevent contamination of the loading buffer. Alternatively,
aliquot 65–70 μl loading buffer into an RNase-free microcentrifuge tube and add 5 μl to each well
from this volume.
3. Pipet 1 µl DNA ladder into the well labeled L. Every chip must have the DNA ladder loaded into the
ladder well labeled L.
4. Pipet 1 µl sample (or blanks, for example water or TE buffer) into sample wells 1–11.
5. Inspect all wells for bubbles by holding the chip above a light-colored background and looking
through the wells (Figure 3.3). Dislodge any bubbles at the bottom of a well with a clean pipet tip
or by removing and reloading the solution.
6. Slide the chip into the Experion vortex station and turn on the vortex station by pressing Mix.
Vortexing continues for 60 sec and then automatically shuts off. Remove the chip when the vortex
station stops.
7. Inspect the wells again to confirm that there is no excessive bubble formation from pipetting, and
that no liquid has spilled outside the wells during vortexing.
8. Place the loaded chip into the Experion electrophoresis station and start the run within 5 min.
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Experion DNA 1K and DNA 12K Analysis Kits
Load 5 µl loading buffer into sample
wells 1–11 and well labeled L
Load 1 µl sample into wells 1 –11
Load 1 µl ladder into well
labeled L
Fig. 3.2. Experion DNA chip. Wells for loading buf fer, samples, GS, and ladder are indicated.
Load 9 µl GS into other 3 wells
labeled GS
Fig. 3.3. Bubble formation during loading of Experion Pro260 chips. Surface bubbles do not generally cause
problems during a run, but bubbles at the bottoms of wells must be removed. Left, bubbles trapped at the bottom of
wells. The GS and G wells and sample wells 1, 3, and 4–6 contain no solution. Wells 8, 10, and L are filled properly and
have no bubbles, but large bubbles have formed at the bottoms of wells 7 and 9 (note the difference in the diameter of
the light-colored circles in wells 8 and 9). Right, bubbles have formed at the surface of the three GS wells on the right
side of the chip; the rest of the wells have no bubbles.
3.7 Run the DNA Analysis
1. Open the lid of the electrophoresis station by pulling the release latch. Place the primed, loaded, and
vortexed chip on the chip platform and close the lid.
2. In the Experion software toolbar, click New Run . In the New Run screen (Figure 3.4), from
the Assay pull-down list, select either DNA > DNA 1K or DNA > DNA 12K.
3. Either select a project folder from the Project pull-down list or create a new project folder by entering
a name in the Project field or by selecting File > Project > New. The project folder appears in the
project tree.
4. Enter a name for the run in the Run Prefix field and click Start Run .
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Experion Automated Electrophoresis System
Fig. 3.4. Details of the New Run scre en. The green dot in the lower right corner indicates that communication
between the electrophoresis station and Experion software has been established.
5. In the New Run dialog (Figure 3.5), select the number of samples to be analyzed. Though all wells
are filled, the Experion system stops the analysis when it reaches the number of samples entered.
Fig. 3.5. New Run dialog. The Experion system stops analysis when it
reaches the number of samples entered.
6. Click Start. The green LED in the center of the front panel on the electrophoresis station blinks, and
the system performs a number of checks: it confirms that a chip has been inserted, that all wells
contain liquid, that electrical connections are made, etc. A calibration counter marks the progress of
these calibrations at the upper right of the screen.
Do not open the lid of the Experion electrophoresis station until the run is complete. The lid does
not lock. Opening the lid aborts the run.
An “IV Check Error” message indicates the system cannot make electrical contact in one of the
wells. This often means there is a bubble at the bottom of the well. Abort the run, and check the
chip for bubbles or empty wells. Refill the affected well(s), and start the run again.
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