Apparatus and method for purification of nucleic acids by phase separation using laser and beads
a technology of phase separation and apparatus, applied in the field of apparatus and method for purification of nucleic acids by phase separation using laser and beads, can solve the problems of loss of cell function, description of a method of disrupting cells, time-consuming and complicated, etc., and achieve the effect of effective purification of nucleic acids
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preparation example 1
[0070]As illustrated in FIG. 1, HBV prepared as described below (60 μl), serum (30 μl) and micro magnetic beads (30 μl, Dynabeads® M-270 Carboxylic Acid, DYNAL, Norway) were mixed in a capillary. 808 nm, 21.1 W high power laser beam (HLU25F100-808, LIMO, Germany) was applied to the mixture for disrupting cells for a designated time in individual experiments while stirring the capillary by vortexing (see FIG. 1).
preparation example 2
HBV, Primer and PCR
[0071]DNA released from the cells was detected using a pair of PCR primers as follows: primer TMP5-F (SEQ ID No: 1); and primer TMP5-R (SEQ ID No: 2). The primer pair was sites corresponding to 2,269-2,387 nucleotides of HBV genome. PCR was performed by 40 cycles (predenaturation at 50° C. for 10 minutes and at 95° C. for 1 minute, denaturation at 95° C. for 5 seconds, and annealing and elongation at 62° C. for 15 seconds) by means of Taq polymerase (Takara, Korea). The amplified DNA was analyzed in Agilent BioAnalyzer 2100 (Agilent Technologies, Palo Alto, Calif.) with a commercially available DNA 500 assay sizing reagent sets.
example 1
Separation of a Magnetic Bead Phase and a DNA Solution Phase
[0072]To investigate a degree of phase separation between a magnetic bead phase and a DNA-containing solution phase according to the form of a container, the degree of phase separation was observed using various types of containers. FIG. 2 shows a container with an inner diameter of 9.95 mm, in which a magnetic bead phase and a DNA solution phase are mixed after laser irradiation. Referring to FIG. 2, when the inner diameter of a container is 9.95 mm, magnetic beads with PCR inhibitors, such as denatured proteins and cell debris, bound thereto are not attached to a glass wall and mixed with the DNA containing solution, so that the magnetic bead phase and the DNA containing solution phase are not separated.
[0073]FIG. 3 shows a container with an inner diameter of 4.88 mm, in which a magnetic bead phase and a DNA solution phase are separated after laser irradiation. A in FIG. 3 shows a container in which the magnetic bead phas...
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