Protocol M.1

Methylation Interference

Solutions

10X T4 Polynucleotide kinase Buffer (-DTT)

700 mM Tris 7.5 700 ml 1M Tris pH 7.5

100 mM MgCl2 100 ml 1M MgCl2

200 ml Q

store at room temperature

10X Annealing Buffer

200 mM Tris 8.0 200 ml 1M Tris pH 8.0

10 mM EDTA 8.0 20 ml 0.5M EDTA pH 8.0

500 mM NaCl 500 ml 1M NaCl

280 ml Q

store at room temperature

DMS Reaction Buffer

50 mM Na cacodylate 8.0 50 ml 1M Na cacodylate pH 8.0

1 mM EDTA 2 ml 0.5M EDTA 8.0

10 mM MgCl2 10 ml 1M MgCl2

938 ml Q

store at -20°

DMS Stop Buffer

1.5M NaOAc 7.0 500 ml 3M NaOAc pH 7.0

1.0M bME 70 ml 14M stock

50 mg/ml tRNA 5 ml 10 mg/ml stock

425 ml Q

store at -20°

 

 

2X EMSA Binding Buffer

20% glycerol 400 ml 50% glycerol

20 mM Tris 7.5 20 ml 1M Tris pH 7.5

100 mM KCl 100 ml 1M KCl

1 mM DTT 2 ml 0.5M DTT

478 ml Q

make fresh for each experiment

Low Salt NET

0.15M NaCl 0.87 g NaCl

20 mM Tris 8.0 2.0 ml 1M Tris pH 8.0

0.1 mM EDTA 8.0 20 ml 0.5M EDTA pH 8.0

98 ml Q

store at room temperature

High Salt NET

1.0M NaCl 5.8 g NaCl

20 mM Tris 8.0 2.0 ml 1M Tris pH 8.0

0.1 mM EDTA 8.0 20 ml 0.5M EDTA pH 8.0

98 ml Q

store at room temperature

20 mM NaPhosphate 7.0

39 ml 1M monobasic

61 ml 1M dibasic

4.9 ml Q

Note: also have 1M NaCl and 1M Hcl on hand

 

 

 

 

 

 

 

 

 

 

Procedure

end-label oligonucleotide probes by kinasing

• Prepare a stock of ss oligonucleotides that are complementary at a concentration of 1.5 mg/ml.

• Combine the following and incubate at 37° for 30':

1 ml each oligo separately

2 ml 10X Kinase Buffer (-DTT)

5 ml g 32P-ATP

1 ml 0.5M DTT

10 ml Q

1 ml Polynucleotide Kinase

• Remove the unincorporated nucleotide by running over a NENSORB column (see protocol S.5) or other appropriate column.

• Dry down and resuspend in 8 ml Q. Add 1 ml of cold complementary oligo and 1 ml 10X Annealing Buffer. Heat to 65° and allow to slow cool on ice in 50 ml of 65° water.

• Add 90 ml cold Q and count 1 ml in the scintilation counter. I usually get 400,000 to 500,000 cpm per ml.

methylate probe

• Combine the following and incubate at room temp. for 5':

5 ml probe (approx. 2 million cpm)

5 ml Q

200 ml DMS Reaction Buffer

2 ml DMS

• Add 40 ml DMS Stop Solution and 600 ml EtOH with 1 ml tRNA. Incubate on dry ice for 5-10' and spin for 15'.

• Wash and dry. Resuspend in 20 ml Q and count 1 ml. I usually get 100,000 cpm per ml.

 

large scale EMSA

• I generally do 50 ml reactions and use the large wells for the gels. Combine the following:

25 ml 2X Binding Buffer

4 ml poly dI/dC (4 mg)

3 ml Probe (300,000 cpm)

15-20 mg Nuclear Extract

Q to 50 ml

• Incubate at room temperature for 30' and run the gel at 4° for 3 hours at 215 Volts. Remove the top plate and wrap the gel and remaining plate in saran wrap. Mark several spots on the gel with fluorescent tape and place an X-ray film with a second glass plate on top of the saran wrap. Expose in a large cassette for 3 hours at 4°.

• Develop the film and place a piece of saran wrap over the film. Mark on the saran wrap the location of the tape and the bands to be cut out. Align this saran wrap over the gel and cut out the bands of interest. Have an 0.8% Agarose gel ready with extra wide lanes and a piece of DEAE NA 40 membrane inserted about 0.5 cm from the wells. Place each gel slice in a well and run the gel at 150mA const. current for 25 minutes. Remove the DEAE membrane and cut off each lane, keep wet in running buffer.

• Wash excess agarose off with 0.5 ml low salt NET by vortexing and spinning in the microfuge. Transfer the membrane to a second eppendorf containing 0.2 ml NET high salt and incubate at 65° for 30 minutes or longer (If the experiment is done in duplicate or triplicate, the membranes can be combined at this step in 0.5 ml NET high salt).

• Remove the NET high salt and wash the membrane with 0.2 ml water, combine with the first sample. Phenol/chloroform extract and precipitate with EtOH and tRNA (No additional saltis required). If the DNA was eluted into 0.5 ml NET high salt there is no need to wash, and the P/C step can be omitted.

 

 

cleavage of the probe with piperidine

• Resuspend the probe in 63 ml Q and 7 ml piperidine. Incubate at 90° for 30 minutes and dry. Resuspend in 30 ml Q and repeat. Resuspend in 20 ml Q and repeat the drying.

cleavage of the probe with NaOH

• Resuspend the probe in 100 ml Q and add100 ml 20mM Na phosphate pH 7.0. Incubate at 90° for 15 minutes and add 20 ml 1M NaOH (while in the heat block). Incubate 30' at 90° and neutralize with 20 ml 1M HCl. Add 25 ml 3M NaOAc and 500 ml EtOH with 0.5 ml tRNA. Incubate on dry ice for 10' and spin for 15'.

• Resuspend in 6 ml formamide loading dye and count. Normalize all samples and run on a 20% denaturing PAGE.

NOTE: The piperidine cleavage gives preferential G cleavage and the NaOH gives both G & A cleavage. Also, I count the whole sample at this point because there aren't many counts. The gel runs best if the amount of tRNA is constant in all the samples, it may be worthwhile to try glycogen.