Template Generation System™ II Kit (TGS™ II Kit)

Transposon-mediated insertion of primer binding sites into target DNA for sequencing or PCR* amplification

Description

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 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. 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
  

System protocol

Day 1. In vitro transposition reaction and transformation Mix Entranceposon, MuA Transposase and target DNA and incubate one hour at 30 °C. During incubation MuA Transposase enzyme catalyses the formation of transposition complex and insertion of the Entranceposon into target DNA at random locations. Heat inactivate at 75 °C for 10 minutes. Transform the reaction mixture, plate and cultivate the cells overnight.
Day 2. Characterization: there are three different options to proceed: a, b, c a. Colony-PCR mapping and DNA sequencing from PCR product Map the Entranceposon insertions in individual clones using colony-PCR and use the resulting PCR products directly as sequencing templates. b. Colony-PCR mapping and plasmid preparation Map the Entranceposon insertions in individual clones using colony-PCR and start overnight liquid cultures from the clones of your choice. Use for colony-PCR primer pairs MuEnd + pUC Fwd and MuEnd+pUC Rev.
c. No mapping, plasmid preparation from randomly selected clones Start overnight liquid cultures from randomly selected clones (especially suitable for target DNA clones that are too large for colony-PCR mapping, e.g. BACs).
Day 3. a. Analyze the DNA sequencing data DNA sequencing using the products from the colony-PCR reactions as template is the fastest way to complete the sequence.
b, c. Plasmid preparation and DNA sequencing Use the primers SeqW or SeqE for sequencing from plasmid DNA.

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Kit components

• Artificial transposons

Entranceposons are re-designed to make transfer of Entranceposon-containing cassettes into other vectors easier. Different antibiotic resistance gene is used in Entranceposon than in the vector to be able to select only clones carrying insertion. TGS II Kit includes now three Entranceposons with three different markers and thus maximize the usability of this kit in different applications.

• F-778 Entranceposon (CamR-3), 20 µl (20 ng/µl in TE buffer)
• F-779 Entranceposon (KanR-3), 20 µl (20 ng/µl in TE buffer)
• F-784 Entranceposon (TetR-3), 20 µl (25 ng/µl in TE buffer)

• F-750 MuA Transposase
20 µl, (0.22 µg/µl in MuA storage buffer)

• F-752 5x Reaction Buffer, 100 µl

• F-753 Control Target DNA
10 µl, (370 ng/µl in TE buffer)

• Primers

• Primers for insertion mapping: (25 µM in dd water)

F-756 MuEnd Primer, 800 µl
MuEnd Primer, binding site at each end of the Entranceposon

F-754 pUC Fwd Primer, 400 µl
F-755 pUC Rev Primer, 400 µl
pUC Fwd, pUC Rev, binding sites in the vector pUC19

• Primers for DNA sequencing: (10 µM in dd water)

F-780 SeqE Primer, 250 µl
F-781 SeqW Primer, 250 µl
SeqE, SeqW, binding sites in the Entranceposon

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Advantages

• 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) and without fragmentation and subcloning
• Simple mapping of the Entranceposon insertions by colony-PCR or by restriction enzyme digestion enables directed sequencing. Consequently, fewer sequencing reactions are required to complete a given 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
• Tree different antibiotic resistance genes available in each TGS II Kit

Ordering information

F-702	Template Generation System™ II Kit (TGS™ II Kit)
The kit contains sufficient materials for 20 reactions
Artificial transposons available separately:
F- 778	Entranceposon (CamR-3)	20 µl (20 ng/µl in TE buffer)
F-779	Entranceposon (KanR-3)	20 µl (20 ng/µl in TE buffer)
F-784	Entranceposon (TetR-3)	20 µl (25 ng/µl in TE buffer)
F-750	MuA Transposase with 5x Reaction Buffer	20 µl, (0.22 µg/µl in MuA storage buffer)

NOTE: TGS™ I Kit still remains available for the customers who have been using it on the regular basis and are requesting for it. Click here for more information about TGS I Kit

Storage stability

Store the components at -20 °C. Stable for 1 year from the date of packaging when stored and handled properly.

Mu Transposition references

Butterfield, Y.S.N. et al. (2002). An efficient strategy for large-scale high-throughput transposon-mediated sequencing of cDNA clones. Nucleic Acids Res. 30, 2460-2468.

Lamberg, A., Nieminen, S., Qiao, M. and Savilahti H. (2002). Efficient insertion mutagenesis strategy for bacterial genomes involving electroporation of in vitro-assembled DNA transposition complexes of bacteriophage mu. Appl. Environ. Microbiol. 68, 705-12.

Vilen, H., Eerikäinen, S., Tornberg, J., Airaksinen, M.S., Savilahti, H. (2001). Construction of gene-targeting vectors: a rapid Mu in vitro DNA transposition-based strategy generating null, potentially hypomorphic, and conditional alleles. Transgenic Res. 10, 69-80.

Haapa, S., Suomalainen, S., Eerikäinen, S., Airaksinen, M., Paulin, L., and Savilahti, H. (1999a). An efficient DNA sequencing strategy based on the bacteriophage Mu in vitro DNA transposition reaction. Genome Res. 9, 308-315.

Haapa, S., Taira, S., Heikkinen, E., and Savilahti, H. (1999b). An efficient and accurate integration of mini-Mu transposons in vitro: a general methodology for functional genetic analysis and molecular biology applications. Nucleic Acids Res. 27, 2777-2784.

Savilahti, H., Rice, P.A., and Mizuuchi, K. (1995). The phage Mu transpososome core: DNA requirements for assembly and function. EMBO J. 14, 4893-4903.

Mizuuchi, K. (1992). Transpositional recombination: mechanistic insights from studies of Mu and other elements. Annu. Rev. Biochem. 61, 1011-1051.