By John M. Walker
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Extra info for New Nucleic Acid Techniques (Methods in Molecular Biology Vol 4)
Example text
16. 17. 10% (w/v) Sodium dithionite in 50 mM sodium hydroxide. Prepare fresh before use. 5% (w/v) sodium nitrite. Prepare fresh, and cool on ice before use. 1M HCl. 0. Deionized formamide. Deionize AR-grade formamide with 1 g/ 10 mL Amberlite MB-3 mixed-bed resin by stirring for 1 h at room temperature. Filter through Whatman No. 1 filter paper and store at -20°C. 1M NaOH. 5M Acetic acid. 0. 4. 4M NaCl. 10% Sodium dodecyl sulfate (SDS). 2% (w/v) SDS, and50% (v/v> formamide. 0. 0. 5M Potassium acetate.
Translation products of RNA with no hybrid arrest. E. Translation products of RNA after hybrid arrest with a control DNA sequence such as actin. F. Molecular weight markers. G. Translation products of RNA after hybrid arrest using just plasmid sequences. References I. 2. 3. 4. 5. 6. 7. 8. 9, Paterson, B. , Roberts, B. , and Kuff, E. , (1977) Structural gene identification and mapping by DNAmRNA hybrid-arrested cell free translation. Proc. Nufl. Ad. Sci USA 74,43704374. Anderson, M. L. M. and Young, B.
6. These hybridization conditions have been used successfully to select some, relatively abundant, tomato mRNAs (9,ZI; Fig. 1). Other tomato fruit cDNA clones have not consistently hybridized sufficient RNA under these conditions to be detectable by translation in vitro. , adjusting the formamide concentration or temperature will alter the stringency, whereas altering the incubation time will determine the extent of hybridization. 7. 5 vol of ethanol and could conceivably be reused for hybridization to other, unrelated DNA papers.