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How does silver staining of DNAwork?


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#1 anonymous

anonymous

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Posted 28 February 2001 - 10:00 PM

I think you can find the protocol you need under the category DNA>Oligos. there are several protocols for this purpose. I myself have tried those protocols. They worked fine.The following is the protocol I used to prepare oligos for bandshift assay

  1. Purify ds oligos with ss oligos by polyacrylamide gel electrophoresis
    1. Prepare by polyacrylamide gel
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      i.  30% polyacrylamide (37.5:1)  10ml

       
       
       
       
      ii.  1X TBE buffer   
       
       
      40ml

       
       
       
      iii.  TEMED  
       
       
      40ul

       
       
       
       
      iv.  APS  
       
       
      400ul

        1. Add 1X loading dye and load all of the sample to the wells and run gel at 150V
        2. Stain gel for 30’ in ethidium bromide.( or in a dark room, place a fluorescent thin-layer chromatographic plate under the gel and examine the gel by illumination from above using a hand-held, long-wavelength UV lamp. The DNA absorbs the UV light and appear dark bands against a uniform fluorescent background)
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        3. Cut band and crush the gel into a slurry. Transfer the slurry into 3 or 4 tubes. Add 1ml  elution buffer (0.1% SDS, 0.5M Ammonium acetate, 10mM Magensium acetate) incubate at 37C with shaking overnight.
        4. Centrifuge the tubes at 12000g for 5min. transfer the pooled supernatants to a 5ml syringe and pass them through a filter (Millipore, 0.45-micron pore size). Collect the effluent in a 15ml tube. ( or filter the gel mixture through a Amicon gel extraction column)
        5. Add 2 vol 100% ethanol, and set at –70C for 30min, centrifuge for 20min and wash with 75% ethanol. Resuspend in TE buffer.
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          If you need any further details, Please email me.





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