Reagent Kits

AATI's capillary electrophoresis instruments utilize specially designed reagent kits to analyze diverse nucleic acid samples, from small RNA to genomic DNA, and NGS library quality control.

Reagent Kit Box

High Sensitivity Large Fragment Analysis Kit

High Sensitivity Large Fragment Analysis Kit
500 Sample High Sensitivity Large Fragment Analysis Kit

The High Sensitivity Large Fragment Analysis Kit is used for the automated sizing and quantification of large DNA fragments and smears. Common applications include as a quality assurance step in long read sequencing, checking the quality of a DNA extraction, and evaluating the quality of degraded DNA samples. Ideal for low concentration samples, this kit is able to size samples through 20 Kb.

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Components

Components 500 Sample Kit 1,000 Sample Kit
Large Fragment Separation Gel 240 mL (DNF-220-0240) 500 mL (DNF-220-0500)
Intercalating Dye 30 µL (DNF-600-U030) 30 µL x 2 (DNF-600-U030)
5X 930 dsDNA Inlet Buffer 125 mL (DNF-355-0125) 300 mL (DNF-355-0300)
5X Capillary Conditioning Solution 50 mL (DNF-475-0050) 100 mL (DNF-475-0100)
HS Large Fragment Diluent Marker Solution 2.4 mL x 5 vials (DNF-381-0003) 2.4 mL x 10 vials (DNF-381-0003)
HS Large Fragment DNA Ladder 100 µL (DNF-379-U100) 100 µL x 2 (DNF-379-U100)
0.25X TE Rinse Buffer 125 mL (DNF-497-0125) 125 mL (DNF-497-0125)
BF-25 Blank Solution 8 mL (DNF-300-0008) 8 mL (DNF-300-0008)

Specifications

Specifications Description
Sample Volume Required 2 µL
Diluent Marker Volume Required 22 µL
DNA Sizing Range 50 bp – 20,000 bp
DNA Sizing Accuracy ± 15% or better
DNA Fragment Concentration Range 5 pg/µL – 0.5 ng/µL input DNA
DNA Smear Concentration Range 50 pg/µL - 5 ng/µL input DNA
DNA Quantification Accuracy ± 25%
DNA Quantification Precision 20% CV
Maximum DNA Concentration 0.5 ng/µL per fragment; 5 ng/µL total
Total Electrophoresis Run Time 50 minutes (Short Array); 80 minutes (Long Array)

Examples

A quality ladder separation is important to the accurate quantitation and sizing of nucleic acid samples. The High Sensitivity Large Fragment DNA Ladder, as shown in Figure 1, provides such a separation when run on the Short Capillary Array (33-55) or the Long Capillary Array (55-80), allowing for the accurate quantification and sizing of large DNA samples. This ladder allows for quantification of fragments down to 0.005 ng/µL and sizing through 20,000 bp.

Figure 1
Figure 1. A typical separation of the High Sensitivity Large Fragment DNA Ladder (DNF-379) on a Fragment Analyzer equipped with a Short Capillary Array (33-55) using the High Sensitivity Large Fragment Analysis Kit (DNF-493).

Through the use of a high quality ladder, separation gel, and electrophoresis parameters, the High Sensitivity Large Fragment Analysis Kit (DNF-493) is able to accurately measure large DNA fragments and smears through 20 Kb. Large DNA fragments are sized with a high degree of accuracy. Figure 2 depicts the separation of a synthetic 15 Kb DNA fragment that was sized at 14.938 Kb, within 1% of the known base pair size.

Figure 2
Figure 2. Separation of a 15 Kb DNA fragment on a Fragment Analyzer equipped with a Short Capillary Array (33-50) using the High Sensitivity Large Fragment Analysis Kit (DNF-493).

Large DNA smear analysis allows for the evaluation of sheared genomic DNA (gDNA) and the assessment of DNA purification quality, among other things. Figure 3 depicts the separation of a 15 Kb DNA smear generated from the shearing of E. coli gDNA. An important feature of PROSize® is the Smear Analysis function which provides an accurate assessment of the smear size distribution. The Smear Analysis region, defined by the dotted red lines, displays the average size of the smear in red.

Figure 3
Figure 3. Separation of sheared E. coli gDNA performed on a Fragment Analyzer equipped with a Short Capillary Array (33-55) using the High Sensitivity Large Fragment Analysis Kit (DNF-493). DNA smears can be analyzed further in PROSize using the Smear Analysis function, indicated by the dotted red lines on the electropherogram. Smear Analysis function sizing is shown in red numbers.

Precise and accurate measurements of large DNA smears and fragments will become increasingly important as molecular biology progresses and new applications develop. Next generation long read sequencing, synthesis of genetic constructs, and restriction digests are only some of the current applications that necessitate accurate measurements of large smears and fragments.

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