Atlas of Plants and Animals in Baltic Amber by Wolfgang; Wichard, Wilfried Weitschat

By Wolfgang; Wichard, Wilfried Weitschat

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The denatured probe should be added directly to the filters in Prehyb buffer, and hybridization carried out for 16–24 h at 42°C. 5. 1% (v/v) SDS at 65°C (100–500 mL/10 filters) for 15 min, with 2 changes of wash buffer. The filters should be wrapped in cellophane paper and exposed to X-ray film for 6–24 h. 6. Using the India ink markings on the filters, the X-rays should be marked, allowing them to be aligned with each original LB plate. 7. Positive colonies from the first hybridization screen should be picked with sterile toothpicks, arrayed on duplicate LB plates (50–100/100-mm-diameter LB plate) and numbered.

RescueMu consists of a plasmid inserted into an intact Mu1 nonautonomous element. RescueMu is inserted downstream of a CaMV 35S promoter in the 5' untranslated leader of maize Lc (Leaf Color) a transcription factor of the R family required for anthocyanin production. Excision of RescueMu can restore tissue pigmentation. Two elements, RescueMu2 and RescueMu3, differ by the presence of unique 400 bp heterologous tags of Rhizobium DNA, and both are present in the original RescueMu transgenic lines.

2. Genomic DNA Isolation (see Note 4) 1. 2. 3. 4. 5. 6. 7. Chloroform. Isoamyl alcohol. Isopropanol. 70% (v/v) Ethanol. Water. 0, 1 mM EDTA. 5/8, 1% (v/v) `-mercaptoethanol, 1% (w/v) sodium bisulfite. For 100 mL of CTAB buffer, dissolve CTAB in 60 mL water by heating in a microwave for 20 s and then add other components. Add `-mercaptoethanol just before use. Store at room temperature or 4°C. 3. Plasmid Rescue 1. Enzymes needed: KpnI, RNaseA, BglII, EcoRI, T4 DNA ligase (Invitrogen). 2. ElectroMAX DH10B competent cells (>1010 colony-forming units [cfu]/µg) (Invitrogen or LIFE Technologies).

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