Our probes are random sheared fragments of DNA made from a variety of sources, cosmid, vector, and even yeast DNA depending upon the nature of the hybridization. The probes are created by taking a random primer and creating a complementary copy of DNA. However, the probe has dioxigenin-11-dUTP which will be incorporated instead of dTTP but will not cause elongation termination like a dideoxynucleotide. The dioxigenin molecule is recognized by the primary antibody.
Sonication and Repair of DNA
1-Pipet 14 µl of dH2O into a 1.5-ml microfuge tube.
2-Pipet 1 µl of DNA into the tube and incubate at 95°C for 10 minutes.
3-Chill sample on ice/NaCl solution for 3 minutes. Keep the Eppendorf on ice.
A solution of NaCl and ice will result in a supercooled solution.
4-Sonicate to shear the DNA.
5-Add 2 µl of hexanucleotide mixture
This is a solution of random hexamers. These will bind to random se quences of the sonicated DNA and serve as markers to start the exten sion.
6-Add 2 µl of dNTP labelling mixture.
This mixture of dNTP is composed of dATP, dCTP, dGTP, and dioxigenin -11-dUTP label which will be inorporated instead of a dTTP. The dioxigenin is the antigenic molecule recognized by the primary antibody.
7-Add 1 µl of Klenow fragment enzyme.
A Klenow fragment is an enzyme that has polymerase activity but no exonuclease activity.
8-Centrifuge briefly and incubate for a minimum of 1 hr (preferably overnight) at 37 °C.
9-Add 2 µl of of 0.2 M EDTA (pH 8.0)
Addition of EDTA inactivates the Klenow fragment.
Recovery of DNA Probe
1-Add 2.5 µl of 3 M sodium acetate (pH 8.0)
2-Add 75 µl of -70 °C Ethanol. Mix well
Addition of NaOAc and EtOH will cause the DNA to precipitate out of solution.
3-Chill tube for a minimum of 30 min at -70 °C or -20 °C overnight.
4-Centrifuge at 12,000 x g for 10 min. Decant.
5-Add 50 µl of chilled 70% EtOH. Centrifuge for 10 min at 12,000 x g.
Removes salts and other contaminants.
6-Decant and dry under vacuum.
7-Resuspend in 50 µl of TE (pH 8.0). Incubate at 37 °C for about 30 min.
TE ( pH 8.0 )