For Real Time Analysis

The MICROLAB STAR is used to fully automate mutant library screens to monitor DNA polymerase reaction in real time with a template reporter molecule based on FRET assay technology. The FRET (fluorescence resonance energy transfer) assay is a very effective tool for monitoring molecule-molecule interactions, functional genomic and proteomic screens, as well as kinetic reactions and enzyme activities. In this application, primary screening of polymerase activity is handled using FRET assay technology.

E.coli lysate from a mutant library is stored cool in a deep well plate, and then placed on the deck of the MICROLAB STAR. The assays are performed in black 384-well reaction plates. First, the reaction mix - consisting of template reporter molecule, primer and dNTPs in buffer solution - is distributed over the reaction plates. Then the reactions are started by adding 5µl of cooled cell lysate from the deep well block. After incubation, the values are read using a fluorescence plate reader.

After hit generation, the hits are characterized in a secondary screening step with radioactive measurement techniques and P32 labelled primers. The Michaelis-Menten kinetics of the enzyme are determined by means of analysis of steady-state kinetics of single nucleotide incorporation. The entire reaction set-up is done in 384-well plates on the MICROLAB STAR and manually evaluated on a gel.

Features and
Benefits

  • System-liquid-free approach permits contamination-free pipetting with radioactive solutions and an economical method of operation, since no waste water is produced and the disposable tips can be disposed of in a sanitary way.
  • Good pipetting precision of MICROLAB STAR, which is necessary in this assay
  • Fast and precise aliquoting of low volumes

System
Configuration

  • MICROLAB STAR, 8 channels
  • H+P shaker for deep well plates, equipped with heating option
  • H+P shaker for deep well plates, equipped with passive cooling option
  • Chilled reagent carrier for tubes and plates, cooled with Haake Kryostat DC-10

Links

Links

www.hp-lab.de
(shaker)

www.thermo.com
(Haake Kryostat)  

 
For Sales and Product information, contact 800-648-5950