PAGE Analysis

Introduction

Polyacrylamide gel electrophoresis (PAGE) offers a high resolution, accurate method for assessment of short DNA fragments. However the process is mostly manual. Samples are loaded into gels, and after electrophoresis a staining application needs to be followed by visualization and fragment analysis.

 

Ranger® Technology automates PAGE

Coastal Genomics has partnered with Hamilton Robotics to fully automate gel electrophoresis for preparative (size selection) and quality control (electrophoretic analysis) purposes. The NIMBUS Select with Ranger Technology is capable of loading polyacrylamide gels, driving electrophoresis, staining gels, imaging (Fig. 1) and assessing fragment lengths.

Figure 1

Fig. 1:  Image of a 10% denaturing PAGE gel that was run and stained by Ranger Technology. Each lane is run with both a 20 and 90 nt marker that Ranger Software uses to assess sample fragment lengths. (1) 10/60 nt Oligo Ladder (IDT), (2)  52 nt fragment, (3) 20/100 nt Oligo Ladder (IDT), (4) 55 nt fragment, (5) 10 – 100 nt Oligo Ladder (Affymetrix), (6) 45 nt fragment.

 

Ranger Analytics offers comprehensive insight

In-lane markers are used by the Ranger Analytics Application to assess single stranded oligo lengths (Fig. 2). This tool can also be used to quantify DNA mass and estimate molarity. Reports can be generated in .csv and .pdf formats.

 

Sizing estimates are highly accurate

Automated sizing of fragments with Ranger Analytics are highly genuine. For single stranded oligos under 100 nt, sizing estimates are typically within 2 nt of the actual length (Fig. 3). This level of accuracy is suitable for characterization of synthetic oligos and other analytes that are sufficiently resolved in polyacrylamide.

Figure 2

Fig. 2: Ranger Analytics GUI is interactive and allows the user to directly compare multiple samples. Traces from lanes (1) and (2) are shown here. Sizing estimates are within 1 nt of the correct value.

Figure 3

Fig. 3: Comparison of Ranger Analytics sizing estimates and actual fragment lengths. Twenty different oligos ranging from 20 to 95 nt in length were run. Some estimates were conducted on oligos that were part of ladder samples (blue) while other samples consisted of a single oligo (red). No estimate was off by more than 2 nt from the actual fragment length.

 

Unparalleled throughput

Together, Ranger Technology and Ranger Analytics enable reliable automation of both polyacrylamide electrophoresis and analysis of resulting images. Urea-based gels are currently available, with native and SDS versions in development.

 

Benefits will include:

  • 384 sample capacity
  • Highly competitive cost per sample
  • <4 hour run time
  • Complete automation from sample load to analysis