Optimum fixed angle centrifuge rotor

Abstract

A centrifuge rotor and method for gradient density separation which supports a generally cylindrical volume of sample solution with its axis inclined to the spin axis at an angle which is optimized for maximum separation efficiency while reducing contamination by undesirable centrifugates upon reorientation of the desirable centrifugates. .Iadd.The sample solution is contained in a centrifuge tube which is closed at its top end by a top portion which is supported by a support cap. The undesirable centrifugates are pelleted to the end corners of the inclined centrifuge tube. The rotor and method are particularly adapted, for example, for separation of nucleic acid into plasmid DNA and chromosomal DNA by density gradient centrifugation. .Iaddend.The optimum angle is determined based on the relationship θ=Tan -1 (D/15L) 0 .5 where D and L are respectively the diameter and length of the cylindrical volume of the sample solution and θ is the angle of inclination.

.[.I.]. .Iadd.We .Iaddend.claim: 
     
       1. A centrifuge rotor comprising: a rotor body rotatable about a spin axis; and   means formed on the rotor body for supporting a generally cylindrical volume of diameter D and length L of sample solution for centrifugation about the spin axis such that the cylindrical volume is inclined with its axis at an angle θ to the spin axis, where θ, D and L approximately satisfy the relationship:   θ=Tan.sup.-1 (D/15L).sup.0.50       
     
     
       2. A centrifuge rotor as in claim 1 wherein θ is approximately 10.45° or less. 
     
     
       3. A centrifuge rotor as in claim 2 wherein θ deviates from the angle calculated from said relationship at given D and L by approximately 14% or less. 
     
     
       4. A centrifuge rotor as in claim 3 wherein θ is approximately 7.5°. 
     
     
       5. A centrifuge rotor as in claim 3 wherein θ is approximately 8.0°. 
     
     
       6. A centrifuge rotor as in claim 3 wherein θ is approximately 9.0°. 
     
     
       7. A centrifuge rotor as in claim 1 wherein the means for supporting comprises the rotor body having a cavity inclined at angle θ to the spin axis and a sample container which is shaped to be received in the cavity. 
     
     
       8. A centrifuge rotor as in claim 7 wherein the sample container is a sealed, generally cylindrical shaped centrifuge tube substantially filled with the sample solution. 
     
     
       9. A centrifuge rotor as in claim 8 wherein the sample solution comprises a density gradient fluid and a sample to be centrifuged by density gradient separation. 
     
     
       10. A centrifuge rotor as in claim 9 wherein the sample is nucleic acid to be separated into at least plasmid DNA and chromosomal DNA isopycnic bands. 
     
     
       11. A centrifuge rotor as in claim 8 wherein the means for supporting further comprises a floating support cap for supporting the top of the centrifuge tube. 
     
     
       12. In a centrifuge rotor for density gradient centrifugation of a generally cylindrical volume of diameter D and length L of sample solution about a spin axis, the cylindrical volume being supported by the rotor such that its axis is inclined at an angle θ to the spin axis where L, D and θ approximately satisfy the relationship:   θ=Tan.sup.-1 (D/15L).sup.0.5     
     
     
       13. A centrifuge rotor comprising: a rotor body defining therein a plurality of cavities distributed axial symmetrically about a spin axis, each cavity having its longitudinal axis inclined at an angle θ to the spin axis; and   at least one container for containing sample solution to be centrifuged, wherein each cavity is shaped to receive the container and the container has an internal space for containing a generally cylindrical volume of sample solution of diameter D and length L, where θ, D and L approximately satisfy the relationship:   θ=Tan.sup.-1 (D/15L).sup.0.5       
     
     
       14. A method of making a centrifuge rotor for density gradient centrifugation comprising the steps of: providing a rotor body rotatable about a spin axis;   forming support on the rotor body for supporting a generally cylindrical volume of diameter D and length L of sample solution such that the axis of the volume is inclined at an angle θ to the spin axis, where D, L and θ approximately satisfy the relationship:   θ=Tan.sup.-1 (D/15L).sup.0.5       
     
     
       15. A method of density gradient centrifugation comprising the steps of: providing a rotor rotatable about a spin axis;   providing a sample solution;   supporting on the rotor a generally cylindrical volume of diameter D and length L of the sample solution such that its axis is nclined at an angle θ to the spin axis, where D, L and θ approximately satisfy the relationship:   θ=Tan.sup.-1 (D/15L).sup.0.5 ; and     rotating the rotor about the spin axis to cause centgrifugation.     
     
     
       16. A method as in claim 15 wherein the supporting step comprises forming a cavity inclined at angle θ in the rotor and providing a sample container which is shaped to be received in the cavity. 
     
     
       17. A method as in claim 16 wherein the sample container provided is a sealed, generally cylindrical shaped centrifuge tube which is substantially filed with sample solution. 
     
     
       18. A method as in claim 17 wherein the sample solution provided comprises a density gradient fluid and a sample to be centrifuged by density gradient separation. 
     
     
       19. A method as in claim 18 wherein the sample is nucleic acid to be separated into at least plasmid DNA and chromosomal DNA isopycnic bands. 
     
     
       20. A method as in claim 15 wherein θ is approximately 10.45° or less. .Iadd. 
     
     
       21.  A centrifuge rotor particularly adapted for density gradient centrifugal separation of a sample comprising: a rotor body having therein a plurality of cavities distributed symmetrically about a spin axis of said rotor body, each cavity having its longitudinal axis inclined at an angle θ to said spin axis and adapted to hold a centrifuge tube which has a cylindrical sidewall for containing a generally cylindrical volume of diameter D and length L of a sample mixture in a density gradient solution to be centrifuged;   said cavities being configured in the rotor body where θ, L and D are such that, upon centrifugation at least first and second pellets of first and second materials are formed at extreme radial corners of the inclined centrifuge tube and extending along the cylindrical sidewall of the centrifuge tube and at least one isopycnic band of a third material is formed vertically in the density gradient solution between the first and second pellets, and upon termination of centrifugation the pellets are stuck to said corners and sidewall of the centrifuge tube and the isopycnic band reorientates into a horizontal orientation with its periphery touching the sidewall of the centrifuge tube but slightly away from the first and second pellets,   whereby the average centrifugal force on said sample mixture is maximized during centrifugation but there is no contact between the third material and either the first and second materials upon horizontal reorientation of the third material upon termination of centrifugation. .Iaddend. .Iadd.22. A centrifuge rotor as in claim 21 wherein θ, D and L approximately satisfy the relationship:   
     
     
       θ=Tan -1  (D/15L) 0 .5 .Iaddend. 23.  A centrifuge rotor as in claim 21 wherein the sample mixture is nucleic acid to be separated into at least plasmid DNA and chromosomal DNA isopycnic bands.  .Iadd.24. A centrifuge rotor as in claim 21 wherein θ is approximately 10.45° or less. .Iaddend. .Iadd.25. A centrifuge rotor as in claim 24 wherein θ is approximately 7.5°. .Iaddend. .Iadd.26. A centrifuge rotor as in claim 24 wherein θ is approximately 8.0°. .Iaddend. .Iadd.27. A centrifuge rotor as in claim 24 wherein θ is approximately 9.0°. .Iaddend. .Iadd.28. A centrifuge rotor as in claim 21 wherein the rotor includes means for supporting a top portion which extends from the cylindrical sidewall of the centrifuge tube and closes top end of the centrifuge tube, said top portion having at its center a tube-like extension through which a sample mixture can be inserted. .Iaddend. .Iadd.29. A method of density gradient centrifugal separation of a sample comprising the steps of: providing a sample mixture in a density gradient solution;   providing a centrifuqe tube which has a cylindrical sidewall for containing a generally cylindrical volume of diameter D and length L of the sample mixture and density gradient solution to be centrifuged;   providing a rotor having therein a plurality of cavities distributed symmetrically about a spin axis of said rotor, each cavity having its longitudinal axis inclined at an angle θ to said spin axis and adapted to hold the centrifuge tube, said cavities being configured in the rotor where θ, L and D are selected such that, upon centrifugation at least first and second pellets of first and second materials are formed at extreme radial corners of the inclined centrifuge tube and extending along the cylindrical sidewall of the centrifuge tube and at least one isopycnic band of a third material is formed vertically in the density gradient solution between the first and second pellets, and upon termination of centrifugation the pellets are stuck to said corners and sidewall of the centrifuge tube and the isopycnic band reorientates into a horizontal orientation with its periphery touching the sidewall of the centrifuge tube but slightly away from the first and second pellets;   placing the centrifuge tube and its contents into the cavity; and   rotating the rotor about the spin axis to effect centrifugal separation of the sample mixture to form at least one isopycnic band of said third material,   whereby the average centrifugal force on said sample mixture is maximized during centrifugation but there is no contact between the third material and either the first and second materials upon horizontal reorientation of the third material upon termination of centrifugation. .Iaddend.   
     
     
        .Iadd.     A method as in claim 29 wherein θ, D and approximately satisfy the relationship:   θ=Tan.sup.-1 (D/15L).sup.0.5 .Iaddend.      .Iadd.31. A method as in claim 29 wherein the sample mixture is nucleic acid to be separated into at least plasmid DNA and chromosomal DNA isopycnic bands. .Iaddend. .Iadd.32. A method as in claim 29 wherein θ is approximately 10.45° or less. .Iaddend. .Iadd.33. A method as in claim 32 wherein θ is approximately 7.5°. .Iaddend. .Iadd.34. A method as in claim 32 wherein θ is approximately 8.0°. .Iaddend. .Iadd.35. A method as in claim 32 wherein θ is approximately 9.0°. .Iaddend. .Iadd.36. A method as in claim 29 wherein the centrifuge tube has a top portion which extends from the cylindrical sidewall of the centrifuge tube and closes top end of the centrifuge tube, said top portion having at its center a tube-like extension through which a sample mixture can be inserted, and wherein the method further including the step of providing means for supporting said top portion of the centrifuge tube after it is placed in the cavity. .Iaddend. .Iadd.37. A method of density gradient centrifugal separation of a nucleic acid sample mixture into at least plasmid DNA and chromosomal DNA isopycnic bands comprising the steps of:   providing a nucleic acid sample mixture in a density gradient solution;   providing a centrifuge tube, said centrifuge tube having a cylindrical sidewall for containing a generally cylindrical volume of diameter D and length L of the sample mixture and density gradient solution to be centrifuged, and a top portion which extends from the cylindrical sidewall of the centrifuge tube and closes top end of the centrifuge tube, said top portion having at its center a tube-like extension through which a sample mixture can be inserted;   providing a rotor having therein a plurality of cavities distributed symmetrically about a spin axis of said rotor, each cavity having its longitudinal axis inclined at an angle θ to said spin axis and adapted to hold the centrifuge tube, said cavities being configured in the rotor where θ, L and D are selected such that, upon centrifugation at least first and second pellets of first and second materials are formed at extreme radial corners of the inclined centrifuge tube and extending along the cylindrical sidewall of the centrifuge tube and at least a plasmid DNA isopycnic band and a chromosomal DNA isopycnic band are formed vertically in the density gradient solution between the first and second pellets, and upon termination of centrifugation the pellets are stuck to said corners and sidewall of the centrifuge tube and said plasmid DNA and chromosomal DNA isopycnic bands reorientate into a horizontal orientation with their peripheries touching the sidewall of the centrifuge tube but slightly away from the first and second pellets;   placing the centrifuge tube and its contents into the cavity;   providing means for supporting said top portion of the centrifuge tube after it is placed in the cavity; and   rotating the rotor about the spin axis to effect centrifugal separation of the sample mixture to form the plasmid DNA and chromosomal DNA bands,   whereby the average centrifugal force on said sample mixture is maximized during centrifugation but there is no contact between the plasmid DNA or chromosomal DNA bands and the first and second pellets upon horizontal reorientation of the third material upon termination of centrifugation.   
     
     
        .Iaddend. .Iadd.38.  A method as in claim 37 wherein θ is approximately 10.45° or less. .Iaddend. .Iadd.39. A method of density gradient centrifugal separation of a sample mixture into at least one isopycnic band comprising the steps of: providing a sample mixture in a density gradient solution;   providing a centrifuge tube, said centrifuge tube having a cylindrical sidewall for containing a generally cylindrical volume of the sample mixture and density gradient solution to be centrifuged, and a top portion which extends from the cylindrical sidewall of the centrifuge tube and closes top end of the centrifuge tube, said top portion having at its center a tube-like extension through which a sample mixture can be inserted;   providing a rotor having therein a plurality of cavities distributed symmetrically about a spin axis of said rotor, each cavity having its longitudinal axis inclined at an angle less than 10.45° to said spin axis and adapted to hold the centrifuge tube;   placing the centrifuge tube and its contents into the cavity;   providing means for supporting said top portion of the centrifuge tube after it is placed in the cavity; and   rotating the rotor about the spin axis to effect centrifugal separation of the sample mixture to form at least one isopycnic band,   whereby the average centrifugal force on said sample mixture is maximized upon centrifugation and mixing of the isopycnic band and other pelleted substance can be avoided upon termination of centrifugation. .Iaddend.   
     
     
        .Iadd.40.  A centrifuge rotor particularly adapted for density gradient centrifugal separation of a sample mixture into at least one isopycnic band, comprising: a rotor body having therein a plurality of cavities distributed symmetrically about a spin axis of said rotor body, each cavity having its longitudinal axis inclined at an angle less than 10.45° to said spin axis and adapted to hold a centrifuge tube which has a cylindrical sidewall for containing a generally cylindrical volume of a sample mixture in a density gradient solution to be centrifuged and a top portion which extends from the cylindrical sidewall of the centrifuge tube and closes top end of the centrifuge tube, said top portion having at its center a tube-like extension through which a sample mixture can be inserted; and   means for supporting said top portion of the centrifuge tube after it is placed in the cavity;   whereby the average centrifugal force on said sample mixture is maximized during centrifugation and mixing of the isopycnic band and other pelleted substance can be avoided upon termination of centrifugation. .Iaddend.