Analysis of plasmid samples on a microchip.
We have developed a LabChip-based plasmid assay that runs on the Agilent 2100 Bioanalyzer. The assay determines the sizes and relative concentrations of the multiple forms of plasmid samples. Twelve samples can be analyzed on each chip in an automated run lasting approximately 30min. By using a supercoiled DNA sizing standard of 2-16kb, the size of the analyzed plasmid can be determined. The resulting MW has a relative standard deviation (CV) <5% and error <5%. Plasmids from 2-8kb can be separated with resolution better than 1kb. Topological isoforms in a plasmid sample can also be separated. However, due to differential staining, the heterogeneity of plasmid samples can only be measured if the signal of each isomer peak can be calibrated with pure standards for every isomer form. For a typical plasmid preparation which predominately is in the supercoiled form, the normalized corrected peak area for the supercoiled form correlates with the plasmid concentration in a broad range of 1-100ng/microl. The measurement is semiquantitative with a CV lower than 20%. A number of applications of this assay on a Labchip will be shown.
Ding
Li
L
Caliper Technologies Corp., 605 Fairchild Drive, Mountain View, CA 94043-2234, USA. lding@arctur.com
Williams
Kathi
K
Ausserer
Walter
W
Bousse
Luc
L
Dubrow
Robert
R
D002623:Chemistry Techniques, Analytical / Q000295:instrumentation / Q000379:methods*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004278:DNA, Superhelical / Q000032:analysis / Q000378:metabolism; D004586:Electrophoresis / Q000379:methods; D008970:Molecular Weight; D010957:Plasmids / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D012984:Software; D013379:Substrate Specificity; D013997:Time Factors
D004278:DNA, Superhelical; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II