, Volume 41, Issue 1, pp 287-307

Improvements of the membrane filter method for DNA:rRNA hybridization

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Abstract

We describe and recommend the following improvements of DNA:rRNA membrane filter hybridization methods. One of our aims was to avoid DNA release from filter discs during hybridization.

  1. 1.

    Our hybridization conditions are 2 SSC in aq. dest., with 20% formamide, 50 C, overnight for 16 hr.

     
  2. 2.

    Duplexing is over in 8–10 hr.

     
  3. 3.

    Formamide has to be very pure (O.D.≤0.2/cm light path at 270 nm).

     
  4. 4.

    RNAase treatment: 250 μg/5 ml 2 SSC/filter at 37 C for 1 hr.

     
  5. 5.

    Our conditions for stepwise thermal denaturation are: 5°C steps from 50C to 90C in 1.5 SSC in 20% formamide.

     
  6. 6.

    Single-stranded DNA, fixed on membrane filters, and stored in vacuo at 4C, can be used reliably for hybridization for up to 20 months.

     
  7. 7.

    Concentrated DNA in 0.1 SSC, quick-frozen at −50 C and stored at −90 C for up to 2 years can be used for hybridization without much change.

     
  8. 8.

    A CsCl gradient purification step yields much purer DNA, but increases the release of DNA from filters by about 20%. Filters with 20% more DNA is a compensation.

     
  9. 9.

    rRNA can be stored for 20 months in SSC or 2 SSC at −12C without changing the hybridization results.