Mutagenesis via Transposition
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Contents
Purpose
This protocol is used to randomly mutate the genes in a bacteria to be used for study
Materials Needed
Protocol
-Using 1 microliter of the EZ-Tn5 (R6KyorilKAN-2) Tnp Transposome, electroporate electrocompetent cells
-Perform electroporation according to the equipment manufacturers recommendations
-Recover the cells immediately after electroporation
-Add SOC medium to the electroporation cuvette to a 1 mL final volume and pipette up and down to mix the cells and medium
-Transfer the mixture to a fresh tube and incubate at 37 C while shaking for 30-60 minutes to allow cell growth
-Plate the cells on 25-50 microgram/mL kanamycin
-The unused cells can be stored at 4 C for up to days for use if more plates need to be streaked
-Extract the genomic DNA from the colonies able to grow on the kanamycin media using the protocol found on Preparation of Genomic DNA from Bacteria
Notes
Papers where this or a similar method has been used
Related Ontology Terms
References
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