DNF-464

High Sensitivity Large Fragment 50 Kb Analysis Kit

High Sensitivity Large Fragment 50 Kb Analysis Kit
500 Sample High Sensitivity Large Fragment 50 Kb Analysis Kit

The High Sensitivity Large Fragment 50 Kb Analysis Kit is used for the automated quality and quantity analysis of large DNA fragments and smears. The inclusion of a 48.5 Kb DNA fragment in the DNA ladder enables the sizing of DNA fragments and smears through 50 Kb. The enhanced sizing range of the kit directly relates to its versatility. It is employed equally well as a quality assurance step for downstream applications such as next-generation long read sequencing.

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Components

500 Sample Kit
Large Fragment Separation Gel 240 mL (DNF-220-0240)
Intercalating Dye 30 µL (DNF-600-U030)
5X 930 dsDNA Inlet Buffer 125 mL (DNF-355-0125)
5X Capillary Conditioning Solution 50 mL (DNF-475-0050)
HS Large Fragment Diluent Marker Solution 2.4 mL x 5 vials (DNF-381-0003)
HS Extended Large Fragment DNA Ladder 125 µL (DNF-365-U125)
0.25X TE Rinse Buffer 125 mL (DNF-497-0125)
Dilution Buffer 1X TE 60 mL (DNF-495-0060)
BF-25 Blank Solution 8 mL (DNF-300-0008)
Eppendorf LoBind® 0.5 mL Tubes Package of 50

Specifications

Specifications Description
Sample Volume Required 2 µL
Diluent Marker Volume Required 22 µL
DNA Sizing Range 75 bp - 48,500 bp
DNA Sizing Accuracy ± 15% or better
DNA Fragment Concentration Range 5 pg/µL – 600 pg/µL input DNA (optimal concentration 500-600 pg/µL)
DNA Smear Concentration Range 50 pg/µL - 5 ng/µL input DNA (optimal concentration of 1 ng/µL)
DNA Quantification Accuracy ± 25 %
DNA Quantification Precision 20 % CV
Maximum DNA Concentration 600 pg/µL per fragment; 5 ng/µL per total sample
Total Electrophoresis Run Time 35 min (Ultra-Short Array, 22-47); 55 min (Short Array, 33-55)

Examples

The High Sensitivity Large Fragment 50 Kb Analysis Kit (HS LF 50 Kb Analysis Kit) is powerful and versatile, able to accurately size DNA fragments and smears from 75 bp through 50 Kb due to the High Sensitivity Extended Large Fragment DNA Ladder. Figure 1 shows the ladder run on a Fragment Analyzer fitted with a standard Short Capillary Array. The extended sizing capability of this kit is due to the 48.5 Kb fragment in the ladder.

Figure 1
Figure 1. High Sensitivity Extended Large Fragment DNA Ladder separated on a Fragment Analyzer fitted with a Short Capillary Array (33-55) using the HS LF 50 Kb Analysis Kit.

A key factor to the versatility of the HS LF 50 Kb Analysis Kit is its ability to quantify and qualify both large DNA smears and fragments. Notably, this kit is recommended for use in the construction of SMRTbell™ large insert libraries by Pacific Biosciences® for post-genomic DNA shearing QC steps, replacing time consuming Pulsed Field Gel Electrophoresis (PFGE). By replacing PFGE with the Fragment Analyzer and the HS LF 50 Kb Analysis Kit (DNF-464), researchers can replace multiple overnight PFGE separations with one hour long separations on the Fragment Analyzer, as shown in Figure 2.

Figure 2
Figure 2. PacBio large-insert library construction workflow overview. FA replaces PFGE to complete critical QC checkpoints in 1 hour (blue boxes). Figure provided by PacBio in Product Note PN: PN101-061316.

PROSize® provides several benefits to the evaluation of SMRTbell libraries, notably the Smear Analysis function and the DNA Quality Number (DQN). Figure 3 depicts the post-separation analysis of a completed SMRTbell library using the Smear Analysis function. The dotted orange lines delineate the region (20,000 bp – 150,000 bp) used in the calculation of the average smear size – 44,392 bp (orange numbers).

Figure 3
Figure 3. Pacific Biosciences SMRTbell library separated using the High Sensitivity Large Fragment 50 Kb Analysis Kit (DNF-464). Electrophoresis performed on a Fragment Analyzer equipped with a Short Capillary Array (33-55). The orange dotted lines demarcate the region to be analyzed by PROSize Smear Analysis function, with the calculated average size shown in orange numbers. The region analyzed was 20,000 bp to 150,000 bp.

DQN is a versatile user-defined quality metric that assists in removing subjectivity from the assessment of sample viability. DQN calculation is a straightforward process, dependent on the user selection of a size threshold. PROSize then measures how much of the sample exceeds the defined threshold and assigns a value between 0 and 10, with a higher value correlating to more of the sample falling above the threshold. Using the same SMRTbell library as Figure 3, Figure 4 depicts the application of DQN. The user-defined threshold of 30,000 bp (DQN30,000) provides a DQN of 7.3.

Figure 4
Figure 4. Pacific Biosciences SMRTbell library separated using the High Sensitivity Large Fragment 50 Kb Analysis Kit (DNF-464). Electrophoresis was performed on a Fragment Analyzer equipped with a Short Capillary Array (33-55). The blue dotted line illustrates the DQN30,000. A 30,000 bp user-selected threshold produces a DQN of 7.3.

DQN can be applied to gauge the success of size selection in a SMRTbell workflow. If a library was size selected at 30,000 bp, a DQN threshold of 30,000 bp should be designated, as all remaining DNA should be larger than 30,000 bp. Therefore, any samples with a DQN 30,000 of less than 10 were not size selected properly.

The High Sensitivity Large Fragment 50 Kb Analysis Kit is well suited to a variety of applications, separating a large DNA smear as easily as a SMRTbell library. Large DNA fragments, such as those needed in the synthesis of large genetic elements, are electrophoresed with superior resolution and accuracy. An example of a large fragment separation is shown in Figure 3 where a 15 Kb fragment was separated on a standard Short Capillary Array.

Figure 5
Figure 5. A synthetic 15 Kb DNA fragment was separated using the High Sensitivity Large Fragment 50 Kb Analysis Kit (DNF-464). Capillary electrophoresis was performed on a Fragment Analyzer equipped with a Short Capillary Array (33-55).

In addition to accurate sizing, the HS LF 50 Kb Analysis Kit provides accurate quantification of DNA. Notably, the concentration large DNA fragments are measured at functionally the same concentration on the Fragment Analyzer using the HS LF 50 Kb Analysis Kit as on a common fluorometric instrument. This relationship is shown in Figure 4 which clearly shows the near one to one relationship between the two methods.

Figure 6
Figure 6. Fragment Analyzer concentrations were plotted against fluorometric instrument concentrations. Samples measured were serial dilutions of a 15 Kb fragment. The same samples were measured for both quantification methods.