METHOD OF PURIFYING NUCLEIC ACID MOLECULES USING PROTEINASE K
The present invention provides methods for purifying nucleic acid molecules, wherein each method includes the steps of: (a) synthesizing nucleic acid molecules in a reaction mixture; (b) contacting the nucleic acid molecules with a proteinase for a period of time sufficient to degrade protein in the reaction mixture; (c) applying the nucleic acid molecules treated in accordance with step (b) to a size-limiting filter so that at least some of the nucleic acid molecules are trapped on the filter; and (d) washing the filter with a phosphate buffer having a pH in the range of from about 5.7 to about 8.5.
1 . A method of purifying nucleic acid molecules, said method comprising the steps of:
(a) synthesizing nucleic acid molecules in a reaction mixture;
(b) contacting the nucleic acid molecules with a proteinase for a period of time sufficient to degrade protein in the reaction mixture;
(c) applying the nucleic acid molecules treated in accordance with step (b) to a size-limiting filter so that at least some of the nucleic acid molecules are trapped on the filter; and
(d) washing the filter with a phosphate buffer having a pH in the range of from about 5.7 to about 8.5.
2 . The method of claim 1 further comprising the step of drying the nucleic acid molecules treated in accordance with step (d) on the filter and resuspending the dried nucleic acid molecules in an aqueous solution.
3 . The method of claim 1 wherein the nucleic acid molecules are cRNA.
4 . The method claim 1 wherein the nucleic acid molecules are cDNA.
5 . The method of claim 1 wherein the nucleic acid molecules are contacted with the proteinase by dissolving a dried proteinase in the reaction mixture.
6 . The method of claim 1 wherein the nucleic acid molecules are contacted with the proteinase by adding an aliquot of proteinase solution to the reaction mixture.
7 . The method of claim 1 wherein the phosphate buffer has a pH in the range of from about 6.0 to about 8.0.
8 . The method of claim 1 wherein the proteinase is selected from the group consisting of proteinase K and Subtilisin A.
9 . The method of claim 8 wherein the proteinase consists essentially of proteinase K.
10 . The method of claim 1 wherein the nucleic acid molecules are contacted with the proteinase for a period of time of between ten minutes and one hour.
11 . The method of claim 1 wherein the nucleic acid molecules are cRNA, the proteinase consists essentially of proteinase K, and the phosphate buffer has a pH in the range of from about 5.7 to about 8.5.
12 . The method of claim 1 wherein the method is automated.