By John N.Abelson, Melvin I.Simon

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Enterica nadB and his auxotrophs in LB. Grow overnight with aeration at 37 . 6 ml of cells. 2 ml of the mixture onto each of 5 L‐Tc plates. Incubate overnight at 37 . 4. Replica print to E‐Tc plates (minimal E glucose), and incubate overnight at 37 . 5% of the TcR recombinants to be linked to either auxotrophic marker, and transduce the recipients to prototrophy. These will grow on the E‐Tc plates. 5. , 1996). 6. Prepare P22‐transducing lysates on each insertion mutant.

If the Mud element is held by growing in the presence of Km, then the cells must maintain the second duplicated segment that carries the wild‐type essential gene and do not show visible segregation (the KmR segregants are not viable) on TTC‐Ara‐Km plates (AraCþ‐only colonies). If the Mud element is inserted into a nonessential gene, KmR segregants are viable, and these cells show visible segregation (the KmR segregants are viable) on TTC‐Ara‐Km plates AraCÀ and AraCÀ/þ. (No Tc), the duplication segregates at high frequency and each colony is AraÀ (red) or has AraÀ (red) sectors.

Insertions defective in carbon or nitrogen source utilization must be screened for according to the specific carbon or nitrogen source. Tn10dTc‐araCþ: Plasmid pDH10 was transformed into Salmonella strain SL4213 (a restriction‐deficient Salmonella strain). , 1984). Transposition recombinants were selected for by selecting for Tn10dTc‐araCþ‐encoded TcR. Tens of thousands of TcR transposition recombinant colonies were pooled, and a P22‐transducing lysate prepared on the randomized pool of insertion mutants.

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Advanced Bacterial Genetics by John N.Abelson, Melvin I.Simon

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