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Thermo Scientific™ Template Generation System II Kit

Catalog No. F702
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F702 20 reactions
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Catalog No. F702 Supplier Thermo Scientific™ Supplier No. F702
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Description

Perform transposon-mediated insertion of primer binding sites into target DNA for sequencing or PCR amplification.

Transposon technology is the first technology that allows scientist to insert constructed linear DNA fragment into circular or linear DNA target randomly and very efficiently. The Thermo Scientific Template Generation System II Kit (TGS II Kit) is based on the transposition reaction of the bacteriophage Mu. This system has been simplified to work in vitro, and an artificial Mu transposon, designated as the Entranceposon, has been constructed. The reaction is catalyzed by a single enzyme, MuA Transposase.

TGS II Kit provides the tools for inserting the Entranceposon into foreign DNA at random locations. Insertion of an Entranceposon into unknown target DNA provides primer binding sites for different applications. A simple and fast in vitro transposition reaction takes place in a single reaction tube.

Highlights

  • Provides templates for DNA sequencing more simply and with less hands-on time than any other method
  • Thousands of ready-to-sequence templates from a single transposition reaction: Enough to sequence even large DNA clones (e.g. BAC clones) without fragmentation or subcloning
  • Simple mapping of the Entranceposon insertions by colony-PCR or restriction enzyme digestion enables directed sequencing. Fewer sequencing reactions are required to complete a sequence than with the shotgun approach
  • Universal primers: The sequencing and mapping primers are included in the kit, no need for custom primers.
  • Bidirectional sequencing: A single template clone can be used for sequencing the flanking DNA on both sides of the Entranceposon insertion
  • Three different antibiotic resistance genes are available in each TGS II Kit

All components needed as well as detailed instruction manual to perform the reactions are included in the kit. TGS II Kit is an updated version of the TGS I Kit.

Specifications

Form Solution
Format Tube
Reaction Speed Fast
Product Type Template Generation System II Kit
Quantity 20 reactions
Sufficient For 20 Reactions
For Use With (Application) Pre-amplification
No. of Reactions 20 Reactions
Starting Material DNA

Frequently Asked Questions (FAQs)

I performed colony-PCR mapping reactions to localize the Entranceposon insertions generated using the TGS Kit and analyzed the amplification products using agarose gel electrophoresis. The PCR product seemed to be a smear rather than an intact band. How do I avoid this problem?

Make sure not to use too much bacteria as a reaction template. Dilute the colony in 50-100 µL water or 0.9% saline and use 1 µL of the dilution per 20 µL PCR reaction. The excess of the E. coli genomic DNA in the PCR reaction typically results in a smeared amplification product. If you use a DNA polymerase from another manufacturer it is likely that the reaction conditions given in the TGS Kit Instruction Manual for the DyNAzyme EXT DNA Polymerase have to be modified.
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 Template Generation System Kit (TGS Kit)?

The Template Generation System Kit provides a method for introducing primer binding sites randomly into foreign target DNA. Insertion of the Entranceposon into foreign DNA makes it possible to sequence the flanking DNA using the primers supplied in the kit. The clones for targeted sequencing can be selected from the population of random insertions by using simple colony-PCR or restriction enzyme mapping.

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