I need a PROTOCOL !!!!

Virginia Urquidi vurquidi at molbiol.ox.ac.uk
Wed Aug 21 07:49:34 EST 1996


In Article <baez-1708960111100001 at dd62-148.compuserve.com>,
baez at chem.columbia.edu (Victor Anaya-Baez) wrote:
>Does anyone have a protocol for Electrophoresis through an agarose gel
>containing methylmercuric hydroxide 

I used to use this protocol, and gave me the best results (sharpest bands). 
I managed to survive the toxicity...but have changed to formamide gels.

PROTOCOL
PREPARING AND RUNNING METHYLMERCURY GELS
                                              '.~
1. Melt a 1% agarose solution (e.g., 160 ml) in 1X EM buffer and
   cool it to 6042oC until used. 
2. Add 750 Ill 1 M methyl mercury solution to the 150 ml of gel
   to obtain a 5 mM final concentration. It is recommended but
   not absolutely necessary (see Chapter 2) that this step and
   all steps up to 14 be performed under a fume hood. 
3. Pour a horizontal gel and insert the comb. 
4. Allow the agarose to solidify; remove the comb. Fill the wells
   with lX EM buffer. Run nonsubmarine gels 
5. Prepare a 1-,ug/,ul RNA solution. 
6. For each sample, mix in a microfuge tube:      
    5 ul RNA solution from step 5     
    5 ,ul 2X sample buffer  
7. Prepare a l/l0 dilution of the 1 M stock solution of methyl
   mercury in sterile distilled water. 
8. Add 1 ul 0.1 M methyl mercury solution to each sample. 
9. Close the caps tightly, vortex, and spin for 1 second at 12000
   rpm.                                       -. 
10. Load samples quickly onto the gel to avoid diffusion. Have
   the gel connected to the power supply and completely set up
   before you begin to load the gel.

1. Run gel at a constant voltage between 60 and 100 V

2. Use a peristaltic pump to recirculate the buffer between
    two tanks. 

13. Place the gel in 200 ml of 0.05 M ammonium sulfate and
    20 ,ul of ethidium bromide solution (10 mg/ml in water)
    Shake the gel for 20 minutes. Dispose the solution.
14.  Repeat step 13. Photograph the gel under UV light.
************************************************
Notes for Methyl Mercury Gels

1. As discussed above and in Chapter 2, working with diluted solutions of
methyl mercury is no more hazardous than work-
ing with several other chemicals currently used in the labo
ratory. We do recommend, however, wearing gloves when ma
nipulating methyl mercury gels and wrapping waste before
discarding.


2. Ammonium phosphate, ammonium sulfate or sodium thio
sulfate will inactivate methyl mercury in case of a spill. 
3. All pieces of gel in which methyl mercury has not been com
plexed should be soaked in a solution of 0.5 M ammonium
phosphate for 30 minutes before being dumped in a waste
basket or wrapped and disposed of as organic waste. 
4. All glasswaue, gel bed, and comb can be rinsed with water
after use, before being processed as regular glassware for
washing. 
5. Since EDTA forms complexes with methyl mercury, it is es
sential to maintain a sufficient excess of methyl mercury to
be denaturing for nucleic acids. EDTA concentration should
6. Chloride ions should not be used in buffers because they also
form complexes with methyl mercury. 
7. There is no need to put methyl mercury in the buffer tanks
for electrophoresis. 
8. 0.1% Xylene cyanol dye can be run as a marker for 18S RNA.
This dye runs just behind 18S RNA in 1% gels. Since the
mobility of the xylene cyanol dye is not affected by the size of
the agarose matrix, the relative migrations of xylene cyanol
and 18S RNA will vary with the agarose concentration in the
gels. 
9. If you wish to run a submarine gel, you should use 10 mM
methyl mercury as diffusion of methyl mercury out of the gel
during a long run may reduce the concentration of the de-
naturing agent below some critical value. A methyl mercury
concentration ranging between 5 and 10 mM is satisfactory
for vertical gels. 
10. Better resolution of small RNA species can be achieved by
running polyacrylamide gels in the presence of methyl mer-
cury (Chandler et al., 1979).

***************************************************

Buffers for Methyl Mercury Gels

All buffers are made with sterile distilled water in baked glassware

EM buffer (l OX)              1 liter

0.05 M Sodium borate       19.0 g Sodium borate.1OH20
0.1 M Sodium sulfate       14.2 g Sodium sulfate
0.01 M EDTA                 3.7 g EDTA
0.15 M Boric acid              30.9 g Boric acid
                                H2O to 1 liter

2X Sample buffer              10 ml

20% Glycerol                  2 ml Glycerol
0.05% Bromphenol blue          6 mg Bromophenol blue
2X EM buffer                   2 ml 10X EM buffer
                                H2O to 10 ml

0.5 M Ammonium acetate

38.55 g Ammonium acetate
H2O to 1 liter
(Store solid powder at 4oC.)

NaOH-mercaptoethanol          400 ml 

                              200 ml H2O
50 m M NaOH                   0.8 g NaOH
5.7 m M 2-Mercaptoethanol     160 ,ul 14.3 M 2-Mercaptoethanol   
                              H2O to 400 ml

Citrate phosphate buffer
(CPB, 0.14 M)                       

13.5 g Citric acid
21.5 g Sodium phosphate
(disodic)- 7H2O
H2O to 1 liter

Citrate phosphate buffer
(CPB,20 m M)
98 ml 140 mM CPB            
H2O to 1400 ml     
                              

REFERENCES                                                            
Bailey, J. M., and Davidson, N. (1976), Anal. Biochem., 70: 75 Chandler, P.
M., Rimkus, D., and Davidson, N. (1979),  
Anal. Biochem., 99, 200. 
Junghans, R. P. (1983), Environ. Res., 31, 1.

Maybe also this: Thomas PS (1980) PNAS 77
*******************************************************************************

Best of luck

V. Urquidi
NDCB-JRH
Oxford
vurquidi at molbiol.ox.ac.uk



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