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Thermo Scientific™ Mutation Generation System Kit

Catalog No. F701
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F701 10 reactions
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Catalog No. F701 Supplier Thermo Scientific™ Supplier No. F701
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Description

Perform functional analysis of proteins with these transposon-based tools.

transposon products are based on the transposition machinery of the bacteriophage Mu. During the lytic phase of the phage's life cycle the machinery replicates its genome by transposing repeatedly inside the host genome. The Mu transposition reaction has been modified into an in vitro reaction catalyzed by a single enzyme - MuA Transposase. In this system, one in vitro reaction is capable of generating more than a million transposon insertion clones.

Highlights

The Mutation Generation System (MGS Kit) and Stop Generation System (STOP Kit) were developed for functional analysis of proteins. These new transposon tools enable the creation of saturated libraries of mutated proteins in a single reaction with less hands-on time than any other method. The location of the transposon insertion in each mutated clone can be mapped by either PCR or sequencing. With MGS and STOP kits, thousands of mutated clones are ready for expression studies in just 2 to 3 days.

The MGS Kit contains the complete set of reagents for transposon-based linker scanning mutagenesis of any target protein. The MGS Entranceposons are designed for making subtle changes in the structure of a target protein by inserting 15 bp in-frame linkers throughout the corresponding target gene. This in-frame insertion allows for conservation of downstream sequences.

The STOPKit Entranceposons contain translational stop codons in all three reading frames within the terminal portion of the transposon sequence. The proprietary modification of the Stop Generation System makes it possible to generate a saturated C-terminal deletion library from virtually any target protein with a maximum addition of three amino acids.

Highlights

  • Efficient - Create saturated insertion libraries for sequencing and protein analysis in a single reaction
  • Fast - Decrease hands-on time compared to conventional methods
  • Random - Eliminate target site preference or insertion hot-spot

Specifications

Form Solution
Format Kit
Reaction Speed Fast
Mutagenesis Capability Mutagenesis at Multiple Sites
Product Type Mutation Generation System Kit
Quantity 10 reactions
Sufficient For 10 Reactions
No. of Reactions 10 reactions
Starting Material DNA

Frequently Asked Questions (FAQs)

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.
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.

For Research Use Only. Not for use in diagnostic procedures.