Agilent Technologies DNA 1000 Kit User manual

Agilent Technologies
Agilent DNA 1000 Kit
Quick Start Guide
Kit Components
Assay Principles
Agilent DNA kits contain chips and reagents designed for sizing and analysis of DNA fragments. Each Agilent
DNA kit contains an interconnected set of microchannels that is used for separation of nucleic acid
fragments based on their size as they are driven through it electrophoretically. Agilent DNA kits are designed
for use with the Agilent 2100 bioanalyzer only.
Assay Kits
The Agilent DNA 1000 kit provides higher resolution of smaller fragments in comparison of our other DNA
sizing kits. It can be used for the following applications:
•Analysis of PCR and RT-PCR products
•RFLP analyses
•Heteroduplex analysis using mismatch cleavage enzymes
•QC of sequencing templates
The complete DNA 1000 kit guide can be found in the online help of the 2100 Expert Software.
Table 1 DNA 1000 Kit
Agilent DNA 1000 Kit (reorder number 5067-1504)
DNA Chips DNA 1000 Reagents (reorder number 5067-1505)
25 DNA Chips (yellow) DNA Ladder
1 Electrode Cleaner (green) DNA Markers (2 vials)
Syringe Kit (blue) DNA Dye Concentrate
1
(1 vial)
1
“This product is provided under a license by Life Technologies Corporation to Agilent Technologies. The purchase of this
product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the
product only as described in accompanying product literature. The sale of this product is expressly conditioned on the buyer
not using the product or its components (1) in manufacturing; (2) to provide a service, information, or data to an unaffiliat-
ed third party for payment; (3) for therapeutic, diagnostic or prophylactic purposes; (4) to resell, sell or otherwise transfer
this product or its components to any third party, or use for any use other than use in the subfields of research and develop-
ment, quality control, forensics, environmental analysis, biodefense or food safety testing. For information on purchasing a
license to this product for purposes other than described above contact Life Technologies Corporation, Cell Analysis Busi-
ness Unit, Business Development, 29851 Willow Creek Road, Eugene, OR 97402, Tel: [phone]. Fax: [phone]."
1 Syringe (red) DNA Gel Matrix (3 vials)
3 Spin Filters

2
Agilent DNA 1000 Kit Quick Start Guide
Storage Conditions
•Keep all reagents and reagent mixes refrigerated at 4 °C when not in use to avoid poor results caused by
reagent decomposition.
•Protect dye and dye mixtures from light. Remove light covers only when pipetting. Dye decomposes when
exposed to light.
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
•0.5 ml microcentrifuge tubes for sample preparation
•Microcentrifuge
Sample Preparation
•PCR samples: For accurate determination of DNA concentration, the total DNA in sample must be
between 0.1–50 ng/μl. If concentration of your particular PCR reaction is excessively high, dilute to
0.1–50 ng/μl.
•Restriction digests: Final concentration of DNA should not exceed 50 ng/μl. Add EDTA and/or heat
inactivate the restriction enzyme according to the manufacturer instructions. Restriction endonucleases
in combination with non-chelated metal ions may degrade internal DNA markers used in assay kit.
Table 2 DNA 1000 Kit Specifications
Physical Specifications Analytical Specifications
Type Specification Type Agilent DNA 1000 Assay
Analysis time 35 min Sizing range 25 – 1000 bp
Number of
samples
12 samples/chip Sizing resolution 25 – 100 bp: 5 bp
100 – 500 bp: 5 %
500 – 1000 bp: 10 %
Sample volume 1 µL Sizing accuracy ± 10 %
Kit stability 4 months (see box for storage
temperatures)
Sizing reproducability 5 % CV
Kit size 25 chips
12 samples/chip
= 300 samples/kit
Quantitation accuracy
1
1Determined using the respective DNA ladder as sample
20 %
Quantitation reproducibility
1
25 – 500 bp: 15 % CV
500 – 1000 bp: 5 % CV
Quantitative range
1
0.5 – 50 ng/µL
Maximum salt concentration in
sample
250 mM for KCl,
15 mM for MgCl
2
,
250 mM NaCl

3
Agilent DNA 1000 Kit Quick Start Guide
Setting up the Chip Priming Station
1
Replace the syringe:
a Unscrew the old syringe from the lid of the chip priming station.
b Release the old syringe from the clip. Discard the old syringe.
c Remove the plastic cap of the new syringe and insert it into the clip.
d Slide it into the hole of the luer lock adapter and screw it tightly to the chip
priming station.
2 Adjust the base plate:
a Open the chip priming station by pulling the latch.
b Using a screwdriver, open the screw at the underside of the base plate.
c Lift the base plate and insert it again in position C. Retighten the screw.
3 Adjust the syringe clip:
a Release the lever of the clip and slide it down to the lowest position.
Essential Measurement Practices
•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.
Agilent DNA Assay Protocol
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 solutions with particular caution as DMSO is known to facilitate the entry of organic
molecules into tissues.

Agilent DNA 1000 Kit Quick Start Guide
*G2938-90015*
*G2938-90015*
G2938-90015 Rev. D
Edition 12/2017
For Research Use Only
Not for use in diagnostic procedures
Printed in Germany
© Agilent Technologies, Inc. 2000-2016, 2017
Agilent Technologies
Hewlett-Packard-Straße 8
76337 Waldbronn, Germany
Preparing the Gel-Dye Mix
1
Allow DNA dye concentrate (blue ) and DNA gel matrix (red ) to equilibrate to room temperature for
30 min.
2 Add 25 μL of DNA dye concentrate (blue ) to a DNA gel matrix vial (red ).
3 Vortex solution well and spin down. Transfer to spin filter.
4 Centrifuge at 2240 g ± 20 % for 15 min. Protect solution from light. Store at 4 °C.
Use prepared gel-dye mix within 6 weeks of preparation.
Loading the Gel-Dye Mix
1
Allow the gel-dye mix to equilibrate to room temperature for 30 min before use.
2 Put a new DNA chip on the chip priming station.
3 Pipette 9 μL of gel-dye mix in the well marked .
4 Make sure that the plunger is positioned at 1 mL and then close the chip priming station.
5 Press plunger until it is held by the clip.
6 Wait for exactly 60 s then release clip.
7 Wait for 5 s, then slowly pull back the plunger to the 1 mL position.
8 Open the chip priming station and pipette 9 μL of gel-dye mix in the wells marked .
Loading the Marker
1
Pipette 5 μL of marker (green ) in all sample and ladder wells. Do not leave any wells
empty.
Loading the Ladder and Samples
Technical Support
Please visit our support web page http://www.agilent.com/genomics/contactus to find information on your
local Contact Center.
Further Information
Visit the 2100 Bioanalyzer site at http://www.agilent.com/genomics/bioanalyzer. You can find useful
information, support and current developments about the products and the technology.
1 Pipette 1 µL of DNA ladder (yellow ) in the well marked .
2 In each of the 12 sample wells pipette 1 µL of sample (used wells) or 1 µL of de-ionized
water (unused wells).
3 Put the chip horizontally in the adapter and vortex for 1 min at the indicated setting
(2400 rpm).
4 Run the chip in the Agilent 2100 Bioanalyzer instrument within 5 min.
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