Agilent Technologies DNA 7500 Manual

Agilent Technologies
Agilent
DNA 7500 and
DNA 12000
Kit Guide

Notices
© Agilent Technologies, Inc. 2000-2015,
2016
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Manual Part Number
G2938-90024 Rev. C
Edition
12/2016
Printed in Germany
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For Research Use Only
Not for use in diagnostic procedures

3
Contents
Contents
1 Agilent DNA 7500 and DNA 12000 Kit 4
2 Equipment Required for a DNA 7500 and DNA 12000 Assay 6
3 Setting up the Assay Equipment and Bioanalyzer 7
Setting up the Chip Priming Station 8
Setting up the Bioanalyzer 9
Vortex Mixer 9
Starting the 2100 Expert Software 10
4 Essential Measurement Practices 11
5 Agilent DNA 7500 and DNA 12000 Assay Protocol 12
Preparing the Gel-Dye Mix 12
Loading the Gel-Dye Mix 14
Loading the Marker 15
Loading the Ladder and the Samples 16
Inserting a Chip in the Agilent 2100 Bioanalyzer 17
Starting the Chip Run 18
Cleaning Electrodes after a Chip Run 20
6 Checking Your Agilent DNA 7500 and DNA 12000 Assay Results 21
DNA 7500 and DNA 12000 Ladder Well Results 21
DNA 7500 and DNA 12000 Sample Well Results 23

4
Agilent Technologies
Agilent DNA 7500 and DNA 12000 Kit 1
Agilent DNA 7500 Kit (reorder number 5067-1506) Agilent DNA 12000 Kit (reorder number 5067-1508)
DNA Chips DNA Chips
25 DNA Chips 25 DNA Chips
1 Electrode Cleaner 1 Electrode Cleaner
DNA 7500 Reagents (reorder number 5067-1507)&
Supplies
DNA 12000 Reagents (reorder number 6067-1509)&
Supplies
(yellow) DNA 7500 Ladder (yellow) DNA 12000 Ladder
(green) DNA 7500 Markers (2 vials) (green) DNA 12000 Markers (2 vials)
(blue) DNA Dye Concentrate
1
(1 vial) (blue) DNA Dye Concentrate
1
(1 vial)
(red) DNA Gel Matrix (3 vials) (red) DNA Gel Matrix (3 vials)
3 Spin Filters 3 Spin Filters
Syringe Kit Syringe Kit
1 Syringe 1 Syringe
1
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Fax: [phone].”

5
Agilent DNA 7500 and DNA 12000 Kit
1
Table 1 Physical Specifications
Type Specification
Analysis time 30 minutes
Samples per chip 12
Sample volume 1 µL
Kit stability 4 months (see box for storage temperatures)
Kit size 12 samples/chip = 300 samples/kit
Table 2 Analytical Specifications
Type Agilent DNA 7500 Agilent DNA 12000
Sizing range 100 – 7500 bp 100 – 12000 bp
Sizing resolution 100 – 1000 bp: 5 %
1000 – 7500 bp: 15 %
100 – 1000 bp: 5 %
100 – 12000 bp: 15 %
Sizing accuracy ± 10 % ± 15 %
Sizing reproducability 5 % CV 5 % CV
Quantitation
accuracy
1
1
Determined using the respective DNA ladder as sample
20 % 25 %
Quantitation
reproducibility
1
100 – 1000 bp: 10 % CV
1000 – 7500 bp: 5 % CV
100 – 1000 bp: 15 % CV
1000 – 12000 bp: 10 % CV
Quantitative range
1
0.5 – 50 ng/µl 0.5 – 50 ng/µl
Maximum salt 250 mM for KCl
15 mM for MgCl
2
250 mM NaCl
250 mM for KCl
15 mM for MgCl
2
250 mM NaCl

6
Agilent Technologies
Equipment Required for a DNA 7500 and
DNA 12000 Assay 2
Equipment Supplied with the Agilent 2100 Bioanalyzer
•Chip priming station (reorder number 5065-4401)
•IKA vortex mixer
Additional Material Required (Not Supplied)
•Pipettes (10 μl, 100 μl and 1000 μl) with compatible tips
•Microcentrifuge tubes:
•0.5 ml for sample preparation
•1.5 ml for gel-dye mix preparation
•Microcentrifuge
Check the Agilent Lab-on-a-Chip webpage for details on assays:
www.agilent.com/chem/labonachip.

7
Agilent Technologies
Setting up the Assay Equipment and
Bioanalyzer 3
Before beginning the chip preparation protocol, ensure that the chip priming
station and the bioanalyzer are set up and ready to use.
You have to
•replace the syringe at the chip priming station with each new kit
•adjust the base plate of the chip priming station
•adjust the syringe clip at the chip priming station
•set up the vortex mixer
•finally, make sure that you start the software before you load the chip.
NOTE
The DNA 7500 and DNA 12000 assay is a high sensitivity assay. Please read this guide
carefully and follow all instructions to guarantee satisfactory results.

8
3 Setting up the Assay Equipment and Bioanalyzer
Setting up the Chip Priming Station
Setting up the Chip Priming Station
1Replace the syringe:
aUnscrew the old syringe from the lid of the chip
priming station.
bRelease the old syringe from the clip. Discard the old
syringe.
cRemove the plastic cap of the new syringe and insert it
into the clip.
dSlide it into the hole of the luer lock adapter and screw
it tightly to the chip priming station.
2Adjust the base plate:
aOpen the chip priming station by pulling the latch.
bUsing a screwdriver, open the screw at the underside
of the base plate.
cLift the base plate and insert it again in position C.
Retighten the screw.
3Adjust the syringe clip:
aRelease the lever of the clip and slide it up to the top
position.
NOTE
Replace the syringe with each new reagent kit.

9
Setting up the Assay Equipment and Bioanalyzer
3
Setting up the Bioanalyzer
Setting up the Bioanalyzer
1Open the lid of the bioanalyzer and make sure that the electrode cartridge is
inserted in the instrument. If not, open the latch and insert the electrode
cartridge.
Figure 1 Electrode cartridge inserted in the instrument (graphic shows an
example).
2Remove any remaining chip.
Vortex Mixer
IKA - Model MS3
1To set up the vortex mixer, adjust the
speed knob to 2400 rpm.

10
3 Setting up the Assay Equipment and Bioanalyzer
Starting the 2100 Expert Software
Starting the 2100 Expert Software
To start the software:
1Go to your desktop and double-click the following icon.
The screen of the software appears in the Instrument context. The icon in the
upper part of the screen represents the current instrument-PC
communication status:
2If more than one instrument is connected to your PC, select the instrument
you want to use in the tree view.
Lid closed, no chip or chip
empty
Lid open
Dimmed icon: no
communication
Lid closed, chip inserted,
DNA or demo assay
selected

11
Agilent Technologies
Essential Measurement Practices 4
•Handle and store all reagents according to the instructions on the label of
the individual box.
•Avoid sources of dust or other contaminants. Foreign matter in reagents
and samples or in the wells of the chip will interfere with assay results.
•Keep all reagents and reagent mixes refrigerated at 4 °C when not in use.
•Allow all reagents and samples to equilibrate to room temperature for
30 min before use.
•Protect dye and dye mixtures from light. Remove light covers only when
pipetting. The dye decomposes when exposed to light and this reduces the
signal intensity.
•Always insert the pipette tip to the bottom of the well when dispensing the
liquid. Placing the pipette at the edge of the well may lead to poor results.
•Use a new syringe and electrode cleaners with each new kit.
•Use loaded chips within 5 min after preparation. Reagents might evaporate,
leading to poor results.
•Do not touch the Agilent 2100 Bioanalyzer during analysis and never place
it on a vibrating surface.

12
Agilent Technologies
Agilent DNA 7500 and
DNA 12000 Assay Protocol 5
After completing the initial steps in “Setting up the Assay Equipment and
Bioanalyzer” on page 7, you can prepare the assay, load the chip, and run the
assay, as described in the following procedures.
Preparing the Gel-Dye Mix
1Allow the blue-capped DNA dye concentrate (blue ) and red-capped DNA
gel matrix (red ) to equilibrate to room temperature for 30 minutes.
WARNING
Handling DMSO
Kit components contain DMSO. Because the dye binds to nucleic acids, it should be
treated as a potential mutagen and used with appropriate care.
➔Wear hand and eye protection and follow good laboratory practices when preparing
and handling reagents and samples.
➔Handle the DMSO stock solutions with particular caution as DMSO is known to
facilitate the entry of organic molecules into tissues.
NOTE
It is important that all the reagents have room temperature before starting the next step.
Protect the dye concentrate from light.

13
Agilent DNA 7500 and DNA 12000 Assay Protocol
5
Preparing the Gel-Dye Mix
2Vortex the blue-capped vial with DNA dye
concentrate (blue ) for 10 seconds and
spin down. Make sure the DMSO is
completely thawed.
3Pipette 25 μl of the blue capped dye
concentrate (blue ) into a red-capped
DNA gel matrix vial (red ). Store the dye
concentrate at 4 °C in the dark again.
4Cap the tube, vortex for 10 seconds. Visually inspect proper mixing of
gel and dye.
5Transfer the complete gel-dye mix to the top receptacle of a spin filter.
6Place the spin filter in a microcentrifuge and spin for 10 minutes at
room temperature at 1500 g ± 20 % (for Eppendorf microcentrifuge, this
corresponds to 4000 rpm).
7Discard the filter according to good laboratory practices. Label the tube
and include the date of preparation.
NOTE
Always use the volumes indicated. Using different volumes in the same ratio will produce
inaccurate results.
NOTE
The prepared gel-dye mix is sufficient for 10 chips. Use the gel-dye within 4 weeks of
preparation.
Protect the gel-dye mix from light. Store the gel-dye mix at 4 °C when not in use for more
than 1 hour.

14
5 Agilent DNA 7500 and DNA 12000 Assay Protocol
Loading the Gel-Dye Mix
Loading the Gel-Dye Mix
1Allow the gel-dye mix to equilibrate to room
temperature for 30 minutes before use. Protect the
gel-dye mix from light during this time.
2Take a new DNA chip out of its sealed bag and place
the chip on the chip priming station.
3Pipette 9.0 μl of the gel-dye mix at the bottom of the
well marked and dispense the gel-dye mix.
4Set the timer to 30 seconds, make sure that the plunger is positioned at
1 ml and then close the chip priming station. The lock of the latch will
click when the chip priming station is closed correctly.
NOTE
Before loading the gel-dye mix, make sure that the base plate of the chip priming station is
in position (C) and the adjustable clip is set to the highest position. Refer to “Setting up
the Chip Priming Station” on page 8 for details.
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NOTE
When pipetting the gel-dye mix, make sure not to draw up particles that may sit at the
bottom of the gel-dye mix vial. Insert the tip of the pipette to the bottom of the chip well
when dispensing. This prevents a large air bubble forming under the gel-dye mix. Placing
the pipette at the edge of the well may lead to poor results.

15
Agilent DNA 7500 and DNA 12000 Assay Protocol
5
Loading the Marker
5Press the plunger of the syringe down until it is
held by the clip.
6Wait for exactly 30 seconds and then release the
plunger with the clip release mechanism.
7Visually inspect that the plunger moves back at
least to the 0.3 ml mark.
8Wait for 5 seconds, then slowly pull back the
plunger to the 1 ml position.
9Open the chip priming station.
10 Pipette 9.0 μl of the gel-dye mix in each of the
wells marked .
Loading the Marker
1Pipette 5 μl of green-capped marker (green )
into the well marked with the ladder symbol
and into each of the 12 sample wells.
SUHVVXUL]H
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NOTE
Protect the gel-dye mix from light. Store the gel-dye mix at 4 °C when not in use for more
than 1 hour.
OPDUNHU
NOTE
Do not leave any wells empty, or the chip will not run properly. Add 5 µl of green-capped
DNA marker (green ) plus 1 µl of deionized water to each unused sample well.

16
5 Agilent DNA 7500 and DNA 12000 Assay Protocol
Loading the Ladder and the Samples
Loading the Ladder and the Samples
1Pipette 1 μl of the yellow-capped ladder vial
(yellow ) in the well marked with the ladder
symbol .
2In each of the 12 sample wells pipette 1 μl
of sample (used wells) or 1 μl of deionized water
(unused wells).
3Set the timer to 60 seconds.
4Place the chip horizontally in the adapter of the IKA vortex mixer and make
sure not to damage the buldge that fixes the chip during vortexing.
5Vortex for 60 seconds at 2400 rpm.
6Refer to the next topic on how to insert the chip in the Agilent 2100
Bioanalyzer. Make sure that the run is started within 5 minutes.
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OVDPSOH
NOTE
For optimal results, samples should be of pH 6 to 9 and should not have an ionic content
greater than twice that of a typical PCR buffer.
CAUTION
Wrong vortexing speed
If the vortexing speed is too high, liquid spill that disturbs the analysis may occur for
samples generated with detergent containing buffers.
➔Reduce vortexing speed to 2000 rpm!

17
Agilent DNA 7500 and DNA 12000 Assay Protocol
5
Inserting a Chip in the Agilent 2100 Bioanalyzer
Inserting a Chip in the Agilent 2100 Bioanalyzer
1Open the lid of the Agilent 2100 Bioanalyzer.
2Check that the electrode cartridge is inserted properly and the chip selector
is in position (1). Refer to “Setting up the Bioanalyzer” on page 9 for details.
3Place the chip carefully into the receptacle. The chip fits only one way.
4Carefully close the lid. The electrodes in the cartridge fit into the wells of
the chip.
5The 2100 expert software screen shows that you have inserted a chip and
closed the lid by displaying the chip icon at the top left of the Instrument
context.
CAUTION
Sensitive electrodes and liquid spills
Forced closing of the lid may damage the electrodes and dropping the lid may cause
liquid spills resulting in bad results.
➔Do not use force to close the lid and do not drop the lid onto the inserted chip.

18
5 Agilent DNA 7500 and DNA 12000 Assay Protocol
Starting the Chip Run
Starting the Chip Run
1In the Instrument context, select the appropriate assay from the Assay menu.
2Accept the current File Prefix or modify it.
Data will be saved automatically to a file with a name using the prefix
you have just entered. At this time you can also customize the file storage
location and the number of samples that will be analyzed.
NOTE
Please note that the order of executing the chip run may change if the Agilent Security Pack
software (only applicable for Agilent 2100 expert software Revision B.02.02 and higher) is
installed. For more details please read the 'User's Guide' which is part of the Online Help of
your 2100 expert software.
NOTE
Run sample numbers can vary between assays.

19
Agilent DNA 7500 and DNA 12000 Assay Protocol
5
Starting the Chip Run
3To enter sample information like sample names and comments, complete
the sample name table.
4Click the Start button in the upper right of the window to start the chip run.
The incoming raw signals are displayed in the Instrument context.
5After the chip run is finished, remove the chip from the receptacle of the
bioanalyzer and dispose of it according to good laboratory practices.
CAUTION
Contamination of electrodes
Leaving the chip for a period longer than 1 hour (e.g. over night) in the Bioanalyzer
may cause contamination of the electrodes.
➔Immediately remove the chip after a run.

20
5 Agilent DNA 7500 and DNA 12000 Assay Protocol
Cleaning Electrodes after a Chip Run
Cleaning Electrodes after a Chip Run
When the assay is complete, immediately remove the used chip from the
Agilent 2100 Bioanalyzer and dispose of it according to good laboratory
practice. Then perform the following procedure to ensure that the electrodes
are clean (i.e. no residues are left over from the previous assay).
1Slowly fill one of the wells of the electrode cleaner with 350 μl deionized
analysis-grade water.
2Open the lid and place the electrode cleaner in the Agilent 2100
Bioanalyzer.
3Close the lid and leave it closed for about 10 seconds.
4Open the lid and remove the electrode cleaner.
5Wait another 10 seconds to allow the water on the electrodes to evaporate
before closing the lid.
NOTE
Use a new electrode cleaner with each new kit.
CAUTION
Leak currents between electrodes
Liquid spill may cause leak currents between the electrodes.
➔Never fill too much water in the electrode cleaner.
NOTE
Replace the used electrode cleaner with each new kit.
NOTE
When switching between different assays, a more thorough cleaning may be required.
For more details please refer to the "Maintenance and Troubleshooting Guide" which
is part of the Online Help of the 2100 Expert software.