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Mutation Generation System Kit - FAQs

View additional product information for Mutation Generation System Kit - FAQs (F701)

5 product FAQs found

Are there any stability problems arising from the fact that the whole Entranceposon is inserted into the target plasmid? Is the Entranceposon capable of further transposition inside host cells? How stable are the target plasmids that carry the Entranceposon?

The Entranceposonshave been designed so that the presence of the MuA Transposase enzyme is an absolute requirement for any transposition activity. The Entranceposons do not contain any genes from the bacteriophage Mu; only the DNA sequences from the right end of the Mu genome that are responsible for the transposase binding. However, the Entranceposons contain >50 bp inverted terminal repeats. To avoid instability resulting from homologous recombination between the repeats, the use of a recA mutant E. coli strain is recommended.

Is there any background problem in the bacteriophage Mu transposition system that is used in your Transposon Products?

No. The Entranceposons that come with the TGS and MGS Kits are non-replicating linear DNA molecules that are not maintained inside E. coli cells.

Is it possible to insert two copies of the Entranceposon in a single target plasmid when using TGS and MGS kits? How can I avoid such double insertions?

By using the optimized in vitro reaction conditions described in the system protocol, the frequency of double insertions is approximately 1% of all the insertion clones.

Is the insertion site selection of the Entranceposon in TGS and MGS kits based on consensus sequence recognition?

Under the optimized reaction conditions of the kits, the naturally occurring consensus sequence preference of the bacteriophage Mu transposition has been minimized. Therefore, the in vitro transposition reaction leads to essentially random insertions of the Entranceposon throughout the target DNA. The plasmid clones in which the Entranceposon insertion destroys either the marker gene conferring resistance to the selective agent or the DNA sequences responsible for the plasmid replication are incapable of amplifying under selective conditions and therefore cannot be isolated from bacterial colonies.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

What can I do with the Mutation Generation System Kit (MGS Kit)?

The Mutation Generation System Kit is designed for rapid construction of insertion mutation libraries from any kind of DNA clones. The system employs the highly efficient transposition machinery of the bacteriophage Mu to generate a pool of 15 bp insertion mutants that can be utilized in various functional analyses of the encoded proteins or regulatory DNA regions.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.