Improvements of the membrane filter method for DNA:rRNA hybridization
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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.
Our hybridization conditions are 2 SSC in aq. dest., with 20% formamide, 50 C, overnight for 16 hr.
Duplexing is over in 8–10 hr.
Formamide has to be very pure (O.D.≤0.2/cm light path at 270 nm).
RNAase treatment: 250 μg/5 ml 2 SSC/filter at 37 C for 1 hr.
Our conditions for stepwise thermal denaturation are: 5°C steps from 50C to 90C in 1.5 SSC in 20% formamide.
Single-stranded DNA, fixed on membrane filters, and stored in vacuo at 4C, can be used reliably for hybridization for up to 20 months.
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.
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.
rRNA can be stored for 20 months in SSC or 2 SSC at −12C without changing the hybridization results.
- Improvements of the membrane filter method for DNA:rRNA hybridization
Antonie van Leeuwenhoek
Volume 41, Issue 1 , pp 287-307
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