模板传代系统 II 试剂盒

Thermo Scientific transposon technology is the first technology that allows scientist to insert constructed linear DNA fragment into circular or了解更多信息

货号	数量
F702	20 reactions

货号 F702

价格（CNY)

20 reactions

请求批量或定制报价

Thermo Scientific 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.

Features:

• 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.

Applications
The Template Generation System II Kit is designed for inserting an artificial Mu transposon into unknown target DNA to provide primer binding sites for:

• Rapid sequencing of templates without primer walking or custom primers
• Shotgun sequencing of large DNA clones directly without time-consuming fragmentation and subcloning into a library
• Insertion of PCR priming sites into target DNA with an unknown sequence
• Random insertional mutagenesis of cloned DNA; direct amplification, mapping or sequencing using primers at the insertion site
• Sequencing with transposons vs. traditional sequencing
• Special Applications:
• BAC sequencing
• Transposition reaction into linear target DNA

仅供科研使用。不可用于诊断程序。

产品规格管装

反应次数20 次反应

产品类型模板传代系统 II 试剂盒

数量20 reactions

原始材料DNA

足够用于20 次反应

适用于（应用）预扩增, Pre-amplification

反应速度快速

Unit SizeEach

常见问题解答 (FAQ)

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.

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

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