Precision fluid gradient formation

Abstract

Ways for reproducibly making liquid gradients with a high degree of precision are provided. Different regions in the gradient are preformed by premixing liquids usable for other components of the gradient to form an intermediate gradient component that is then added to the vessel. The system is particularly adapted for making non-linear and multiple overlapping different gradients in the same liquid in the same vessel.

What is claimed is:  
     
         1 . A gradient component kit containing components of a liquid gradient comprising at least three liquids, one having a lower level gradient component liquid, one having a higher level gradient component liquid and one having an intermediate level gradient component liquid, wherein the intermediate level gradient component liquid is prepared by mixing the lower level gradient component liquid and the higher level gradient component liquid.  
     
     
         2 . The gradient component kit of  claim 1  wherein the gradient component liquids have different densities.  
     
     
         3 . The gradient component kit of  claim 1  wherein the gradient component liquids have different concentrations.  
     
     
         4 . The gradient component kit of  claim 1  wherein the gradient component liquids contain different chemical or physical features.  
     
     
         5 . The gradient component kit of  claim 1  wherein at least one of the gradient component liquids contains a readily detectable component capable of determining the location of said gradient component in the liquid gradient.  
     
     
         6 . The gradient component kit of  claim 1  wherein at least one of the gradient component liquids contains a non-sedimenting reagent capable of reacting with a sample passing through the gradient component liquid.  
     
     
         7 . The gradient component kit of  claim 1  further comprising a secondary intermediate level gradient component liquid wherein the secondary intermediate level gradient component liquid is prepared by mixing the intermediate level gradient component liquid with another gradient component liquid.  
     
     
         8 . The gradient component kit of  claim 1  further comprising a series of containers or a multi-welled container wherein each gradient component is located in a different container or a different well.  
     
     
         9 . The gradient component kit of  claim 1  containing gradient components for forming two different gradients simultaneously  
     
     
         10 . A method for forming a liquid gradient comprising; 
 preparing an intermediate level gradient component liquid by mixing a lower level gradient component liquid with a higher level gradient component liquid to, and    layering a first gradient component liquid on a second gradient component liquid wherein a least one of said first and second gradient components is the intermediate level gradient component liquid.    
     
     
         11 . The method of  claim 10  wherein the level gradient component liquids have different densities.  
     
     
         12 . The method of  claim 10  wherein the level gradient component liquids have different concentrations.  
     
     
         13 . The method of  claim 10  wherein the level gradient component liquids contain different chemical or physical features.  
     
     
         14 . The method of  claim 10  further comprising preparing a secondary intermediate level gradient component by mixing the intermediate level component with a different gradient component liquid, and wherein the secondary intermediate level component is one of the gradient components in the liquid gradient.  
     
     
         15 . A liquid gradient produced by the method of  claim 10 .  
     
     
         16 . The liquid gradient of  claim 15  wherein the gradient is an unstable non-linear gradient containing a region containing a reagent.  
     
     
         17 . A vessel containing the liquid gradient of  claim 15 .  
     
     
         18 . The method of  claim 10  wherein the liquid gradient is non-linear.  
     
     
         19 . The method of  claim 10  wherein the liquid gradient is a step gradient.  
     
     
         20 . The method of  claim 10  further comprising adding a sample to the top of the liquid gradient and sedimenting the sample through at least part of the liquid gradient.  
     
     
         21 . The method of  claim 10  further comprising forming a second type of gradient within the liquid gradient.  
     
     
         22 . An apparatus for preparing a gradient in a vessel comprising, 
 a vessel for holding said gradient,    a plurality of containers or a multi-welled container wherein different gradient components are located in different containers or different wells,    a pipette with means for aspirating liquid into and dispensing liquid from the pipette,    a movable aligning device capable of moving the pipette horizontally and vertically with respect said vessel and said plurality of containers or multi-welled container thereby positioning the pipette inside said plurality of containers or multi-welled container and above said vessel,    
     
     
         23 . The apparatus of  claim 22  wherein said plurality of containers or multi-welled container contain at least three gradient components and one gradient component is prepared by mixing two other gradient components.  
     
     
         24 . The apparatus of  claim 22  wherein said movable aligning device is a movable stage holding said vessel and/or said plurality of containers or multi-welled container  
     
     
         25 . The apparatus of  claim 22  wherein said movable aligning device is an arm holding said pipette.  
     
     
         26 . The apparatus of  claim 23  wherein said gradient is a density gradient.  
     
     
         27 . The apparatus of  claim 23  wherein said gradient is a concentration gradient.  
     
     
         28 . The apparatus of  claim 22  further comprising a removable pipette tip attached to a nozzle end of said pipette.  
     
     
         29 . The apparatus of  claim 22  further comprising a second pipette, a fluid line between the pipette and said second pipette, and a pump means for transferring liquid aspirated from the pipette to the second pipette where it is dispensed.  
     
     
         30 . The apparatus of  claim 29  wherein the second pipette is positioned so that it dispenses liquid at the bottom of the vessel below the level of liquid in the vessel.  
     
     
         31 . The apparatus of  claim 22  wherein the vessel is a sector tube.  
     
     
         32 . The apparatus of  claim 23  wherein two different gradients are formed in said vessel.  
     
     
         33 . A gradient component kit containing components of a liquid gradient comprising at least two liquids, one having a higher level of gradient component and one having a lower level of gradient component wherein at least one of the gradient component liquids contains a non-sedimenting reagent capable of reacting with a sample passing through the gradient component liquid.  
     
     
         34 . The gradient component kit of  claim 33  further comprising a third liquid gradient component having a level of gradient component, and 
 wherein the gradient component containing the reagent has a level of gradient component between that of the third liquid gradient component and another liquid gradient component.  
 
     
     
         35 . A method for forming a liquid gradient comprising; 
 adding a first gradient component into a vessel,    adding a second gradient component into a vessel, and    adding a third gradient component into a vessel,    wherein the second gradient component contains a non-sedimenting reagent capable of reacting with a sample passing through the second gradient component.    
     
     
         36 . The method according to  claim 35  wherein the second gradient component material also has a level of gradient component between that of the first gradient component and the third gradient component.  
     
     
         37 . An apparatus for creating density gradients comprising; 
 a robotic pipette apparatus configured to deliver solutions to and from a plurality of tubes, and    a computer controller for controlling said pipette apparatus, said controller adapted for receiving information identifying a plurality of density gradient layers, said controller being connected to said pipette apparatus for operating said pipetter to produce the identified plurality of density gradient layers in one of the plurality of tubes.    
     
     
         38 . The apparatus of  claim 37  wherein said pipette apparatus includes; 
 a base,  
 a first track supported on said base,  
 a first guide supported on said first track for horizontal movement along said first track,  
 a second track supported on said first guide,  
 a second guide supported on said second track for horizontal movement generally perpendicular to said first track, and  
 a mechanically controlled pipette supported by said second guide for vertical movement with respect thereto.  
 
     
     
         39 . A method for computer implemented production of a density gradient, comprising the steps of; 
 specifying the initial solutions from which a density gradient is to be made,    calculating intermediate solutions required to produce the specified layers of the density gradient,    producing the calculated intermediate solutions, and    moving, solution by solution, the intermediate solutions into a density gradient tube.    
     
     
         40 . The method of  claim 39 , further comprising the step of repeating all necessary steps to reproducibly produce multiple density gradients in a plurality of density gradient tubes.