DEVICE AND METHODS FOR BIOLISTIC TRANSFORMATION
The present invention provides an innovate method and device for depositing coated particles to prepare sample cartridges for use in a gene gun. It allows for scalability of sample cartridge preparation, as well as the ability to prepare multiple different samples simultaneously thereby reducing the time required to prepare different samples and consequently the time required to perform a biological assay, such as a transformation as
1 . A method for simultaneously depositing particles on the interior surfaces of a plurality of separate pieces of tubing, the method comprising:
a) preparing a suspension of particles coated with a biological substance in an evaporable liquid;
b) introducing the particle suspension into each separate piece of tubing of the plurality of tubing pieces; and
c) simultaneously passing a gas through each piece of tubing via a manifold to dry the particles onto the interior surface if each of the tubing pieces, the manifold comprising: i) an elongated lumen defining a central fluid flow pathway; and ii) a plurality of fluid connector ports disposed along the lumen, wherein each piece of tubing is in fluid connection with a fluid connector port whereby the gas is allowed to simultaneously flow from the lumen into each piece of tubing, thereby simultaneously depositing particles on the interior surface of each of the pieces of tubing.
2 . The method of claim 1 , wherein (b) comprises:
coupling the syringe to an end of a piece of tubing; and
drawing the suspension of particles into the piece of tubing using the syringe.
3 . The method of claim 1 , wherein a different suspension of particles is introduced into two or more separate pieces of tubing.
4 . The method of claim 3 , wherein the particles of each different suspension are coated with a different biological substance.
5 . The method of claim 4 , wherein the biological substance comprises one or more nucleic acid molecules.
6 . The method of claim 5 , wherein the biological substance comprises at least one DNA molecule and at least one RNA molecule.
7 . The method of claim 1 , further comprising rotating the tube about its longitudinal axis to distribute the particles on the interior surface of the tubing.
8 . The method of claim 1 , with the proviso that the method does not include continuous rotation of each tube while (b), (c) or both (b) and (c) are performed.
9 . The method of claim 1 , wherein each fluid connector port optionally comprises a valve to regulate gas flow into each piece of tubing.
10 . The method of claim 1 , wherein each piece of tubing is less than about 12, 11, 10, 9, 8, 7, 6, 5 or 4 inches in length.
11 . The method of claim 1 , wherein each piece of tubing is about 5-7 inches in length.
12 . The method of claim 1 , wherein at least 2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12 or more separate pieces of tubing are utilized.
13 . The method of claim 12 , wherein each separate piece of tubing comprises a different suspension of particles, each different suspension comprising a different biological substance.
14 . An apparatus comprising:
a) a manifold comprising: i) an elongated lumen defining a central fluid flow pathway; and ii) a plurality of fluid connector ports disposed along the lumen; and
b) two or more pieces of tubing,
wherein each of the two or more pieces of tubing is in fluid connection with a fluid connector port of the manifold.
15 . The apparatus of claim 14 , wherein the interior surface of at least one piece of tubing of the two or more pieces of tubing is deposited with a particle coated with a different biological substance than coats a particle deposited in at least one other piece of tubing of the two or more pieces of tubing.
16 . A method of coating particles with at least one DNA molecule and at least one RNA molecule, comprising:
providing a slurry of metal particles in a solution of spermidine and at least one DNA molecule;
adding calcium chloride to the slurry of metal particles;
pelleting the metal particles;
resuspending the particles in aqueous solution;
adding RNA to the particles in aqueous solution;
alcohol precipitating the RNA and metal particles; and
resuspending the particles in alcohol to provide particles coated with DNA and RNA.
17 . The method of claim 16 , wherein the particles are gold or tungsten.
18 . The method of claim 16 wherein alcohol precipitating is precipitating with ethanol or isopropanol.
19 . A preparation of particles of particle bombardment of cells, wherein the particles are coated with at least one RNA molecule and at least one DNA molecule.
20 . A preparation of particles of particle bombardment of cells, wherein the particles are coated with at least two RNA molecules and at least one DNA molecule.