Analytical specifications
1,2
dsDNA 130 assay
DNA Sizing Range
75 bp – 20,000 bp
DNA Sizing Accuracy2
+ 10% or better
DNA Sizing Precision2
5% CV
DNA Fragment Concentration Range1
75 – 1,500 bp < 10%, 1,500 – 20,000 bp ≤ 15%
Physical Specifications
Total electrophoresis run time
33cm: 30 minutes, 55cm: 75 minutes
Samples per run
96-Capillary: 95 (+1 DNA Ladder Well) or 96 (Imported DNA Ladder)
Sample volume required
2 µL
Kit stability
4 months
Agilent ZAG-130 dsDNA Kit
Quick Guide for ZAG
DNA Analyzer Systems
The Agilent ZAG DNA Analyzer system is an automated capillary electrophoresis platform for scalable,
flexible, fast, and reliable electrophoresis of DNA fragments.
This Quick Guide is intended for use with the Agilent ZAG DNA Analyzer system only. The ZAG-130 dsDNA kit is
designed for analyzing double-stranded DNA fragments from 75 to 20,000 base pair.
Specifications
0.5 ng/µL – 50 ng/µL input DNA (adjustable by dilution sample)
Separation Resolution
1
Results using DNA ladder in 1X TE buffer.
2
Results using DNA samples in 1X TE buffer.
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ZAG-130 dsDNA Quick Guide for ZAG DNA Analyzer Systems
concentration in 1X TE Buffer
Qualitative DNA,
1000/5000, RT
DNF
100mL
FS
WARNING
•
•
Kit Components – 5000 Sample Kit
Kit Component
Number
Part Number
(Re-order Number)
Description Quantity Per Kit
5191-6610*
ZAG-130-FR*
5191-6615*
ZAG 130/135 dsDNA Kit, 5000, 4oC
ZAG-130-0500 ZAG 130 dsDNA Separation Gel, 500mL 1
DNF-495-0125 Dilution Buffer 1x TE, 125mL 1
DNF-355-0500 5x 930 dsDNA Inlet Buffer, 500 mL
• Dilute with sub-micron filtered water prior
to use
ZAG 130 dsDNA, FR
DNF-600-U030
FS-SLR930-U100
FA-SMK930-0003 75 bp and 20,000 bp Markers, 3.2 mL 1
-475-0100
-SMO15
Intercalating Dye, 30 μL
1,000 Plus DNA Ladder, 100µL
• 75 bp – 20,000 bp; 50 ng/µL total DNA
• 0.5 ng/µL concentration each in 1x TE
buffer
5x Capillary
Conditioning Soln,
Mineral Oil Dropper
Bottle, 15mL
1
1
1
2
2
*Not orderable
Refer to product safety data sheets for further information
When working with the ZAG DNA Analyzer kit components follow the appropriate safety
procedures such as wearing goggles, safety gloves and protective clothing.
2
ZAG-130 dsDNA Quick Guide for ZAG DNA Analyzer Systems
Ambient operating temperature: 19 – 25 °C (66 – 77 °F)
Ensure that no sample or Diluent Marker remains within or on the outside of the tip
• Apply a new seal to 96-well sample plate prior to mixing and centrifugation
suggested to perform one of the following methods to ensure complete mixing.
Additional Material Required for Analysis with the ZAG DNA Analyzer Systems
• ZAG DNA Analyzer system with LED fluorescence detection:
• ZAG DNA Analyzer system (p/n M5320AA)
• ZAG 96-Capillary Array Short, 33 cm (p/n A2300-9650-3355) OR
• ZAG 96-Capillary Array Long, 55 cm (p/n A2300-9650-5580):
• Agilent ZAG DNA Analyzer controller software (Version 1.0 or higher)
• Agilent ProSize Data Analysis software (Version 2.0.0.61 or higher)
Additional equipment/reagents required (not supplied)
• 96-well PCR sample plates. Please refer to Appendix – ZAG DNA Analyzer Compatible Plates and Tubes in the ZAG DNA
Analyzer System User Manual for a complete approved sample plate list
• Multichannel pipettor(s) and/or liquid handling device capable of dispensing 1 – 100 µL volumes (sample plates) and
1,000 µL volumes (inlet buffer plate)
• Pipette tips
• 96-well plate centrifuge (for spinning down bubbles from sample plates)
• Sub-micron filtered DI water system (for diluting the 5x 930 dsDNA Inlet Buffer and 5x Capillary Conditioning Solution)
• 96-deepwell 1mL plate: Fisher Scientific #12-566-120 (inlet buffer and/or waste plate)
• Reagent reservoir, 50 mL (VWR #89094-680 or similar) (for use in pipetting inlet buffer plates/sample trays)
• Conical centrifuge tubes for prepared separation gel/dye mixture and/or 1x Capillary Conditioning Solution
• 250 mL conical: Corning #430776, available from Fisher Scientific #05-538-53 or VWR #21008-771
• Vortexer (for mixing of samples, ladders, and/or markers in tubes and/or plates)
Capillary Storage Solution (p/n GP-440-0100)
•
Essential Measurement Practices
Environmental
conditions
Steps before
sample preparation
Pipetting practice
Mixing and
centrifugation
recommendations
•
• Keep reagents during sample preparation at room temperature
• Allow reagents to equilibrate at room temperature for 30 min prior to use
• Pipette reagents carefully against the side of the 96-well sample plate or sample
tube
•
• When mixing sample with Diluent Marker (DM), it is important to mix the contents of
the well thoroughly to achieve the most accurate quantification. It is highly
3
ZAG-130 dsDNA Quick Guide for ZAG DNA Analyzer Systems
After mixing, briefly centrifuge and visually confirm that all liquid is collected at the
plate
1 (1 FC Only)
2.5 µL
25 mL
40 mL
2 (1 FC +1 GP)
3.0 µL
30 mL
80 mL
5 (1 FC + 4 GP)
4.5 µL
45 mL
120 mL
8 (1FC + 7GP)
6.0 µL
60 mL
160 mL
10 (1 FC + 9 GP)
7.5 µL
75 mL
200 mL
FC=Full Conditioning
GP = Gel Prime Only
bottom of the 96-well sample plate or tube strips and any air bubble is removed
• After adding 2 µL of sample or ladder to the 22 µL of 1x TE, place a plate seal
on the sample plate and vortex the sample plate at 3,000 rpm for 2 min. Any
suitable benchtop plate vortexer can be used. Ensure that there is no well-towell transfer of samples when vortexing. The plate should be spun via a
centrifuge after vortexing to ensure there are no trapped air bubbles in the
wells.
• After adding 2 µL of sample or ladder to the 22 µL of 1x TE, use a separate
pipette tip set to a larger 20 µL volume, and pipette each well up/down to
further mix.
• Use an electronic pipettor capable of mixing a 10 µL volume in the tip after
dispensing the 2 µL sample or ladder volume. Some models enable using the
pipette tip for both adding and mixing.
• Run samples immediately after preparation, or within a day with oil overlay. If not
using right away, cover and keep at 4°C, warm to RT and centrifuge before running
Gel preparation
Prepare gel/dye mixture for ZAG DNA Analyzer System. To ensure the gel/dye mixture is mixed homogeneously without generating bubbles, gently invert the centrifuge tube 5 to 10 times, depending on the volume of the mixture.
ZAG DNA Analyzer system volume specifications for 96-capillary
# of 96-well plates
to be Analyzed
1
Volume of Intercalating
Dye
Volume of Separation
2
Gel
Volume of 1x Conditioning
2
Solution
4
ZAG-130 dsDNA Quick Guide for ZAG DNA Analyzer Systems
•
•
Follow the appropriate safety procedures such as wearing goggles, safety gloves and protective clothing.
ZAG system - 96 capillary; Ladder – well H12
Agilent ZAG 130 dsDNA assay operating procedure
1. Mix fresh gel and dye according to the volumes in the Gel preparation tables. Refill 1x Capillary Conditioning
Solution as needed.
2. Place a fresh 1x 930 dsDNA Inlet Buffer in drawer ‘B’ on the system, 1.0 mL/well. Replace daily.
2.1. ZAG system - 96 capillary; Fill all rows of buffer plate
3. Prepare Capillary Storage Solution plate. Replace every 2-4 weeks for optimal results.
3.1. ZAG system - 96 capillary; Fill all rows of a sample plate with 100 µL/well, place in drawer ‘S’
4. Place Marker plate in drawer ‘M’ on the system, 30 µL/well with 30 µL overlay (one drop) of
Mineral Oil.
4.1. ZAG system - 96 capillary; Fill all rows of sample plate
5. Dilute 1 kb Plus DNA Ladder (50 ng/µL) to desired working concentration:
5.1 Dilute 12x (2 µL ladder per 22 µL 1x TE buffer) for samples > 10 ng/µL.
5.2 Dilute 50x (1 µL ladder per 49 µL 0.1x TE buffer) for samples < 10 ng/µL.
6. Mix samples with Diluent Buffer 1X or 0.1x TE depending on the concentration. Place prepared
ladder in ladder well.
WARNING
Working with Chemicals
The handling of reagents and chemicals might hold health risks.
Refer to product material safety datasheets for further chemical and biological safety information.
5
ZAG-130 dsDNA Quick Guide for ZAG DNA Analyzer Systems
Agilent ZAG DNA Analyzer software operating procedure
1. Select Row, Group or Tray to run.
2. Enter sample ID and Tray ID (optional).
3. Select Add to Queue, from the dropdown menus select the corresponding method based on your capillary length;
3.1 ZAG130FC33 – DNA 75-20000bp – Full Conditioning
3.2 ZAG130GP33 – DNA 75-20000bp – Gel Prime Only
3.3 ZAG130FC55 – DNA 75-20000bp – Full Conditioning
3.4 ZAG130FP55 – DNA 75-20000bp – Gel Prime Only
4. Enter Tray Name, Folder Prefix, and Notes (optional).
5. Select OK to add method to the queue.
6. Select to start the separation.
1,000 bp DNA Ladder result
Representative 1,000 bp Plus DNA Ladder result injected with 75 bp lower marker and 20,000 bp upper marker, using the ZAG
system with the ZAG-130-5000 reagent kit. Method:
ZAG130FC33 (short array).
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ZAG-130 dsDNA Quick Guide for ZAG DNA Analyzer Systems
Issue
Cause
Corrective Action
The peak signal is >> 20,000 RFU;
1 Input DNA sample concentration is
1 Dilute input DNA sample
130)
No peak observed for DNA sample
1 Sample concentration too low and
1 Prepare more concentrated sample
and mixed in sample well.
Sample peak(s) migrate before or
1 Excess primer-dimer species in
1 Further dilute input DNA sample
primer-dimer species.
Sample peak(s) migrate after of
Marker.
1 DNA sample size out of range of
1 Fragment samples if possible and
Poor resolution of ladder peaks.
1 Capillary Array Vent Valve is partially
1 Inspect and if necessary clean
Troubleshooting
The following table lists several potential assay specific issues which may be encountered when using the ZAG 130
dsDNA kit (75-20000 bp) and suggested remedies. Contact Agilent technical support if you have any additional
troubleshooting or maintenance questions.
upper marker peak is low or not
detected relative to lower marker.
when expected. Lower/Upper
Marker peaks observed.
co-migrate with 75 bp Lower
Marker
too high.
out of range
2 Sample was not added to 1x TE
diluent or not mixed well
sample
concentration with 1x TE buffer and
repeat experiment; OR
Repeat experiment using decreased
injection time (e.g., 10 sec); OR
Prepare fresh sample using ZAG
130 dsDNA (1-500 bp) (Part # ZAG-
and repeat experiment. (e.g. 4 µL +
20 µLDI Water) OR Repeat
experiment with increased injection
time and/or injection voltage for
Marker and Sample Plates.
2 Verify sample was correctly added
concentration with 1x TE buffer to
minimize primer-dimer interference
and repeat experiment.
2 If fragment size is below 5,000 bp,
analyze using ZAG-110 dsDNA Kit
(35 bp – 5,000 bp) to better resolve
co-migrate with 20,000 bp Upper
Slower migration time than
expected.
assay.
plugged with gel.
reanalyze.
Capillary Array Vent Valve as
described in the ZAG
Troubleshooting and Maintenance
Guide.
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ZAG-130 dsDNA Quick Guide for ZAG DNA Analyzer Systems
Technical Support and Further Information
For technical support, please visit www.agilent.com. It offers useful information, support and current developments about the
products and technology.
www.agilent.com
© Agilent Technologies, Inc. 2020
Edition 10/20
SD-AT000147
8
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