Cleaning and liquid contact with solids – Apparatus – With means for collecting escaping material
Reexamination Certificate
2002-05-10
2004-08-17
Stinson, Frankie L. (Department: 1746)
Cleaning and liquid contact with solids
Apparatus
With means for collecting escaping material
C134S201000, C366S273000, C366S274000
Reexamination Certificate
active
06776174
ABSTRACT:
BACKGROUND AND SUMMARY OF THE INVENTION
The present invention relates generally to life sciences applications, and more particularly to an apparatus for efficient washing of magnetic particles in molecular biology/immunology applications. One such application is cDNA molecule isolation.
One approach for cDNA clone isolation requires the use of streptavidin-coated magnetic particles (approximately 1 &mgr;m in size) to capture biotinylated-oligo cDNA hybrids, which are subsequently made double stranded and transformed into a cell such as
E. coli
. A time consuming portion of this approach is the cDNA Capture procedure. During the cDNA Capture procedure, extensive washing of the streptavidin-coated magnetic particles are performed after the capture of the cDNA molecules. See generally The GeneTrapper™ cDNA Positive Selection System Instruction Manual, Life Technologies Inc., which is incorporated herein by reference in its entirety.
Conventionally, the cDNA Capture procedure uses six 1.7 ml tubes and a six-position magnet to attract the magnetic particles to one side of each of the 1.7 ml tubes. During the cDNA Capture procedure, the nucleic acid containing mixture (comprising a cDNA library hybridized to biotinylated-oligonucleotides) is mixed with the streptavidin-coated paramagnetic beads by gently pipetting. The suspension is incubated for 30 minutes at room temperature. During this process, the suspension is gently mixed every two to three minutes by finger tapping or vortexing at the lowest speed for ten seconds to re-suspend the beads. The tubes are then inserted into the magnet for two minutes and the supernatant is removed and discarded. Finally, the beads are extensively washed.
The extensive washing procedure consists of adding 100 &mgr;l of wash buffer to the beads, re-suspending the beads by finger tapping or gently vortexing at the lowest speed, and re-inserting the tubes into the magnet for two minutes. The supernatant is removed and discarded, and this process is repeated.
An additional 100 &mgr;l of wash buffer is added to the beads. The beads are resuspended by pipetting. The solution is then transferred to clean tubes and the clean tubes are inserted into the magnet for five minutes. The supernatant is removed and discarded and 100 &mgr;l of wash buffer is immediately added to each tube. Each tube is then finger tapped or vortexed and the tubes are again inserted into the magnet for five minutes. After the five minute incubation, the supernatant is removed and discarded, and 20 &mgr;l of 1× elution buffer is added to the beads and mixed by pipetting. The beads are incubated for five minutes at room temperature. During the incubation, the beads are finger tapped for ten seconds every minute. The tubes are then inserted into the magnet for an additional five minutes. The supernatant (which now contains the captured cDNA molecules) is transferred and saved in fresh tubes.
Throughout the above defined processes, the tubes are inserted into the magnet and, after a 2-5 minute wait period, the supernatant is removed from the tube and a solution (TE buffer, wash buffer, or elution buffer) is added to the tube by pipetting. Each 1.7 ml tube is then handled separately in order to re-suspend the paramagnetic beads after they have been pulled to one side of the tube by the magnet. This process requires picking up each tube individually and agitating the tube by finger tapping or gently vortexing the tube at the lowest speed to avoid splashing beads to the sides of the tube. The tube is then re-inserted into the magnet for an additional 2-5 minutes and the supernatant is then removed.
This process, which is repeated a number of times throughout the procedure for cDNA molecule isolation, is time consuming and can take up to approximately 45 minutes for each tube. Also, the process of finger tapping permits inconsistencies and variability between tubes.
An efficient means for simultaneously washing magnetic particles in a plurality of tubes, without finger tapping or vortexing, that renders consistent test results is needed.
The present invention satisfies the above mentioned needs by providing an apparatus that can efficiently wash multiple tubes, preferably 96 tubes, simultaneously within 5 minutes without having to individually finger tap or vortex the tubes. As will be evident, the present invention also provides high throughput operation as well as automation for any application which utilizes magnetic particles, particularly magnetic or paramagnetic beads.
The present invention is an apparatus for washing magnetic or paramagnetic particles, such as might be useful in life sciences applications, including molecular biology/immunology applications. The present invention includes a well plate which preferably has a substantially flat upper surface, a substantially flat lower surface, and a plurality of wells uniformly placed in rows and columns. Each of the wells has an opening in the upper surface of the well plate that preferably tapers conically to a lower end. The upper surface of the well plate preferably has a recessed area extending around the perimeter and through the first and last rows of the plurality of wells for receiving an array tube holder, preferably a 96 array tube holder. According to a preferred embodiment, the array tube holder can be placed in one of two positions, i.e. a first position and a second position. The array tube holder holds tubes, preferably 0.2 ml tubes, containing a solution comprising magnetic or paramagnetic particles. The lower surface of the well plate preferably includes an asymmetrical arrangement of a plurality of slots positioned in rows and columns. The first row of slots is preferably placed between an edge of the well plate and the first row of wells. Subsequent rows of slots are placed between the remaining rows of wells.
The present invention further comprises a plurality of magnets inserted into each slot in the lower surface of the well plate. Each magnet is positioned adjacent to the lower end of a well for attracting the magnetic particles in the tube toward one side of the tube. In each adjacent row of tubes, the magnetic particles are attracted to the opposite side of the tubes. A base plate is preferably attached to the lower surface of the well plate for securing the magnets.
The present invention may be used to wash or move the magnetic particles by attracting the magnetic particles to one side of the tube by placing the array tube holder in either the first position or the second position and pipetting a washing solution into each tube. The array tube holder is then shifted to the other of the two positions to reverse the magnetic field. Alternatively, the magnetic field can be reversed by changing the position of the tubes, such as by rotating the tubes, preferably by rotating the tubes 180°. The magnetic field can also be reversed by moving the magnets, such as by shifting the magnets between the rows of wells or by changing the position of the magnets, preferably by flipping the magnets, more preferably by flipping the magnets 180°. The reversal of the magnetic field causes the magnetic particles to stream across the tube in the washing solution, preferably in a widest part of the tube, in response to the magnetic attraction of the magnet on the other side of the tube, thereby efficiently washing the magnetic beads in a short time, preferably about five seconds. The supernatant is removed and discarded and the process of reversing the magnetic field and washing the beads is preferably repeated, more preferably, the process is repeated about three times.
Further features and advantages of the invention, as well as the structure and operation of the invention, are described in detail below with reference to the accompanying drawings.
REFERENCES:
patent: 3750243 (1973-08-01), Prentice
patent: 3787034 (1974-01-01), Shvartsman et al.
patent: 3848363 (1974-11-01), Lovness et al.
patent: 3869251 (1975-03-01), Tsantker et al.
patent: 3892908 (1975-07-01), Lovness
patent: 3995835 (1976-12-01), Cichy et al.
pat
Li Wu Bo
Nisson Paul E.
Schuette Michael W.
Sterne Kessler Goldstein & Fox P.L.L.C.
Stinson Frankie L.
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