METHODS FOR REMOVING NUCLEIC ACID CONTAMINATION FROM REAGENTS
In general, the disclosed method can be used to remove contaminating microbes and nucleic acids from microorganisms-derived reagents, apparatus and processes (materials and apparatus) related to PCR (and RT-PCR), including sample prep reagents and materials that are used to isolate, purify and detect nucleic acids.
1 . A method for removing nucleic acid contamination in a PCR master mix reagent comprising:
a. adding a nuclease to the PCR master mix reagent;
b. incubating the PCR master mix reagent plus nuclease; and
c. inactivating the nuclease.
2 . The method of claim 1 , wherein the PCR master mix is formulated for amplifying microorganism nucleic acid.
3 . The method of claim 1 , wherein the nuclease is a DNase or an RNase.
4 . The method of claim 1 , wherein the inactivation of the nuclease is by heat.
5 . The method of claim 1 , wherein the nuclease is bound to a solid surface.
6 . The method of claim 5 , wherein the solid surface is a bead.
7 . The method of claim 6 , wherein the bead is selected from a group consisting magnetic, non-magnetic, glass, and cellulose beads.
8 . The method of claim 6 , further comprising removal of the nuclease by centrifugation, filtration or magnetic separation.
9 . A method for removing DNA contamination in a solution comprising:
a. passing the solution through a column packed with immobilized DNase coated beads or a tube internally coated with DNase; and
b. collecting a DNase treated solution from the column or the tube.
10 . A method for removing RNA contamination in a solution comprising:
a. adding RNase to the solution;
b. incubating the solution with RNase; and
c. inactivating the RNase.
11 . The method of claim 10 , wherein the RNase is bound to a solid surface.
12 . The method of claim 11 , wherein the solid surface is a bead.
13 . The method of claim 11 , wherein the bead is selected from a group consisting magnetic, non-magnetic, glass, and cellulose beads.
14 . The method of claim 11 , further comprising removal of the RNase by centrifugation, filtration or magnetic separation.
15 . A method for removing microorganism contamination in a PCR master mix comprising:
a. sonicating the PCR master mix;
b. heating the PCR master mix at 70° C.;
c. adding DNase to the PCR master mix;
d. incubating the PCR master mix reagent at 37° C.; and then
e. incubating the PCR master mix reagent at 75° C.
16 . The method of claim 15 , further comprising:
centrifuging the PCR master mix to pellet the microorganism contamination;
removing the PCR master mix supernatant from the pellet; and
transferring the supernatant to a clean vessel.
17 . The method of claim 15 , wherein the DNase is immobilized on a solid surface.
18 . The method of claim 15 , wherein the incubating at 37° C. is between at least 5 minutes and at least 30 minutes.
19 . The method of claim 15 , wherein the incubating at 75° C. is for at least 8 minutes.
20 . The method of claim 15 , wherein the heating at 70° C. is for at least 20 minutes.
21 . The method of claim 17 , wherein the solid surface is selected from the group consisting of an insoluble matrix, a glass bead, a magnetic bead, a non-magnetic bead, a tube and a column.
22 . The method of claim 17 , further comprising removal of the DNase by centrifugation, filtration or magnetic separation.