By John N.Abelson, Melvin I.Simon
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Extra info for Advanced Bacterial Genetics
Every mutation should be assigned a name that corresponds to a particular gene or phenotype, and an allele number that identifies each specific isolate. When available for a particular group of bacteria, genetic stock centers are the ultimate resources for gene names and allele numbers. ca/kesander/), and the E. edu/). , 1996). ’’ Due to the absence of clear rules for naming genes, each investigator assigned new names based on the method of isolation, which often resulted in the same name being applied to different genes or different names being applied to the same gene.
Transfer to a new tube and incubate at 37 with shaking for 30 to 60 min. 6. Plate cells on LB agar containing 50 g/ml of kanamycin. Select KanR colonies overnight. 7. Miniprep plasmid DNA using standard techniques. 8. Sequence the rescued clones using the forward and reverse EZ‐Tn5
Rescue Cloning 1. Prepare genomic DNA from your clone of interest using standard or kit‐based techniques. 2. Digest 1 g of genomic DNA using a restriction endonuclease that cuts near the end of the EZ‐Tn5 transposon, but avoids restriction of the 20 transposons  kanamycin resistance gene, the R6K
ori, or the region between these two elements. Several restriction sites are available and allow for optimization of clone size dependent on the GC content of the target genome. 3. 1 to 1 mole of DNA and two units of T4 DNA ligase in a standard reaction for 10 min at room temperature.
Advanced Bacterial Genetics by John N.Abelson, Melvin I.Simon