Sunday, October 19, 2003

Description
Uracil Interference Assay

Procedure
1. Label the 5' end of the oligonucleotide primers with [32P] (CAUTION See Hint #1) and T4 Polynucleotide Kinase. The oligonucleotides should flank the protein-binding site and prime for the opposite strands.

2. Prepare two parallel PCR reaction as follows and run 8 cycles (or more for the right amount of

material) of an appropriate PCR program for your fragment of interest:

5 l of DNA fragment containing the protein binding site (0.2 pmol),

5 l of one [32P]-labeled oligonucleotide primer (20 pmol),

5 l of the other oligonucleotide primer (unlabeled, 20 pmol),

5 l of the 2 mM dNTP mix, 5 l of 10X Taq Polymerase Buffer,

19 l ddH2O, 5 U Taq Polymerase.

3. Run the PCR product on nondenaturing PAGE (see appropriate protocol).

4. Extract the PCR product from the gel by Phenol extraction (see phenol extraction of DNA from polyacrylamide gel protocol). Precipitate the DNA by adding an equal volume of Ethanol and 2.0 M Ammonium Acetate to 0.2 M. Centrifuge at high speed in a microcentrifuge for 15 min to pellet the DNA. Remove the supernatant. Resuspend the pellet in 70% Ethanol then repellet the DNA and remove the Ethanol.

5. Measure the radioactivity (in cpm) of the DNA pellet. Dissolve the DNA in TE buffer at approximately 20,000 cpm/l.

6. Combine protein sample and radiolabeled PCR product in a DNA-binding reaction. Use approximately 105 cpm of DNA probe in 50 l.

7. Run the DNA sample on a native polyacrylamide gel (see appropriate protocol).

8. Autoradiograph gel and remove the corresponding bands and purify DNA from gel slices (see appropriate protocol).

9. Resuspend the DNA in 25 l TE buffer.

10. Remove the Uracil from the DNA in the following reaction: 19 l ddH2O, 5 l 10X Taq Polymerase Buffer, 25 l DNA from step 9, 1 U uracil-N-Glycosylase. Incubate at 37C for 1 hr. Add 100 % Ethanol to stop the reaction and centrifuge in a microcentrifuge at maximum speed for 15 min and save the pellet.

11. Resuspend the pellet in 100 l of 1M Piperidine. Incubate at 90C for 30 min.

12. Lyophilize the samples for 1 hr or until completely dry.

13. Repeat step 12 once.

14. Count the samples and determine the cpm.

15. To the pellet add stop/loading dye. Heat the sample to 90C for 5 min and quickly chill samples in an ice bath.

16. Run samples on a 6 to 8 % sequencing gel and locate free probe and bound complex (see sequencing gel protocol).

17. Autoradiograph the gel.


Recipes
70 % Ethanol

2 mM dNTP mix 2 mM dCTP
2 mM dGTP
2 mM dATP
2 mM dTTP


0.2M Sodium Acetate

1M Piperidine

TE Buffer 10 mM Tris-HCl, pH 7.5-8.0
1 mM EDTA


1M Tris-HCl, pH 8.4 10 mg/ml Gelatin
5 M KCl
50 mM MgCl2


Stop/loading dye prepare in deionized Formamide
20 mM EDTA
0.05 % Xylene Cyanol FF
0.05 % (w/v) Bromophenol Blue



Supplies


Tips
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.