Improvements of the membrane filter method for DNA:rRNA hybridization.
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. Our hybridization conditions are 2 SSC in aq. dest., with 20% formamide, 50 C, overnight for 16 hr. 2. Duplexing is over in 8-10 hr. 3. Formamide has to be very pure (O.D. less than or equal to 0.2/cm light path at 270 nm). 4. RNAase treatment: 250 mug/5 ml 2 SSC/filter at 37 C for 1 hr. 5. Our conditions for stepwise thermal denaturation are: 5 degrees C steps from 50 C to 90 C in 1.5 SSC in 20% formamide. 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. 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. A CsCl gradient purification step yields much purer DNA, but increases the release of DNA from filters by about 20%. Filters with 20% more DAN is a compensation. 9. rRNA can be stored for 20 months in SSC or 2 SSC at -12 C without changing the hybridization results.
D001173:Arthrobacter / Q000032:analysis; D002499:Centrifugation, Density Gradient; D002586:Cesium / Q000378:metabolism; D002861:Chromobacterium / Q000032:analysis; D004247:DNA / Q000302:isolation & purification*; D004355:Drug Stability; D004356:Drug Storage; D005374:Filtration / Q000379:methods; D009693:Nucleic Acid Hybridization / Q000187:drug effects; D012335:RNA, Ribosomal / Q000302:isolation & purification*; D012231:Rhizobium / Q000032:analysis; D012260:Ribonucleases / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors
D012335:RNA, Ribosomal; D002586:Cesium; D004247:DNA; D012260:Ribonucleases