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Droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling for simpler and faster PCR assay using wire-guided manipulations.

You DJ, Yoon JY - J Biol Eng (2012)

Bottom Line: Following extraction, the 1500 bp sequence of Peptidase D from E. coli was amplified using rapid droplet thermocycling, which took 10 min for 30 cycles.The results of these sequentially executed processes were analyzed using gel electrophoresis.Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of accuracy and automation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Agricultural and Biosystems Engineering, The University of Arizona, Tucson, AZ 85721-0038, USA. jyyoon@email.arizona.edu.

ABSTRACT
A computer numerical control (CNC) apparatus was used to perform droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling on a single superhydrophobic surface and a multi-chambered PCB heater. Droplets were manipulated using "wire-guided" method (a pipette tip was used in this study). This methodology can be easily adapted to existing commercial robotic pipetting system, while demonstrated added capabilities such as vibrational mixing, high-speed centrifuging of droplets, simple DNA extraction utilizing the hydrophobicity difference between the tip and the superhydrophobic surface, and rapid thermocycling with a moving droplet, all with wire-guided droplet manipulations on a superhydrophobic surface and a multi-chambered PCB heater (i.e., not on a 96-well plate). Serial dilutions were demonstrated for diluting sample matrix. Centrifuging was demonstrated by rotating a 10 μL droplet at 2300 round per minute, concentrating E. coli by more than 3-fold within 3 min. DNA extraction was demonstrated from E. coli sample utilizing the disposable pipette tip to cleverly attract the extracted DNA from the droplet residing on a superhydrophobic surface, which took less than 10 min. Following extraction, the 1500 bp sequence of Peptidase D from E. coli was amplified using rapid droplet thermocycling, which took 10 min for 30 cycles. The total assay time was 23 min, including droplet centrifugation, droplet DNA extraction and rapid droplet thermocycling. Evaporation from of 10 μL droplets was not significant during these procedures, since the longest time exposure to air and the vibrations was less than 5 min (during DNA extraction). The results of these sequentially executed processes were analyzed using gel electrophoresis. Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of accuracy and automation.

No MeSH data available.


Related in: MedlinePlus

Serial dilution protocol for E. coli sample A-H). Wire-guided syringe needle manipulates, splits, and mixes sample on a hydrophobic surface. Mixing performed with a vibration motor.
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Figure 2: Serial dilution protocol for E. coli sample A-H). Wire-guided syringe needle manipulates, splits, and mixes sample on a hydrophobic surface. Mixing performed with a vibration motor.

Mentions: Figure 2 (and Additional file1) illustrates a representative algorithm of the serial dilution protocol, in which an initial 20 μL droplet of PBS was mixed with 2 μL of E. coli cultured in LB broth, and serially diluted 4 times. Standard plate counting revealed consistent dilutions attributed to the accuracy of the linear actuator in conjunction with the 1 mL disposable syringe needles. Clearing the surface for the next set of procedures was as simple as either extracting the droplets and then dumping the pipette tip, or simply guiding the droplet off of the surface into a KimWipes to be absorbed and later discarded.


Droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling for simpler and faster PCR assay using wire-guided manipulations.

You DJ, Yoon JY - J Biol Eng (2012)

Serial dilution protocol for E. coli sample A-H). Wire-guided syringe needle manipulates, splits, and mixes sample on a hydrophobic surface. Mixing performed with a vibration motor.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3526397&req=5

Figure 2: Serial dilution protocol for E. coli sample A-H). Wire-guided syringe needle manipulates, splits, and mixes sample on a hydrophobic surface. Mixing performed with a vibration motor.
Mentions: Figure 2 (and Additional file1) illustrates a representative algorithm of the serial dilution protocol, in which an initial 20 μL droplet of PBS was mixed with 2 μL of E. coli cultured in LB broth, and serially diluted 4 times. Standard plate counting revealed consistent dilutions attributed to the accuracy of the linear actuator in conjunction with the 1 mL disposable syringe needles. Clearing the surface for the next set of procedures was as simple as either extracting the droplets and then dumping the pipette tip, or simply guiding the droplet off of the surface into a KimWipes to be absorbed and later discarded.

Bottom Line: Following extraction, the 1500 bp sequence of Peptidase D from E. coli was amplified using rapid droplet thermocycling, which took 10 min for 30 cycles.The results of these sequentially executed processes were analyzed using gel electrophoresis.Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of accuracy and automation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Agricultural and Biosystems Engineering, The University of Arizona, Tucson, AZ 85721-0038, USA. jyyoon@email.arizona.edu.

ABSTRACT
A computer numerical control (CNC) apparatus was used to perform droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling on a single superhydrophobic surface and a multi-chambered PCB heater. Droplets were manipulated using "wire-guided" method (a pipette tip was used in this study). This methodology can be easily adapted to existing commercial robotic pipetting system, while demonstrated added capabilities such as vibrational mixing, high-speed centrifuging of droplets, simple DNA extraction utilizing the hydrophobicity difference between the tip and the superhydrophobic surface, and rapid thermocycling with a moving droplet, all with wire-guided droplet manipulations on a superhydrophobic surface and a multi-chambered PCB heater (i.e., not on a 96-well plate). Serial dilutions were demonstrated for diluting sample matrix. Centrifuging was demonstrated by rotating a 10 μL droplet at 2300 round per minute, concentrating E. coli by more than 3-fold within 3 min. DNA extraction was demonstrated from E. coli sample utilizing the disposable pipette tip to cleverly attract the extracted DNA from the droplet residing on a superhydrophobic surface, which took less than 10 min. Following extraction, the 1500 bp sequence of Peptidase D from E. coli was amplified using rapid droplet thermocycling, which took 10 min for 30 cycles. The total assay time was 23 min, including droplet centrifugation, droplet DNA extraction and rapid droplet thermocycling. Evaporation from of 10 μL droplets was not significant during these procedures, since the longest time exposure to air and the vibrations was less than 5 min (during DNA extraction). The results of these sequentially executed processes were analyzed using gel electrophoresis. Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of accuracy and automation.

No MeSH data available.


Related in: MedlinePlus