Transposon Products, FAQ

Q 01: What can I do with the Template Generation System™ Kit (TGS™ Kit) ?
The Template Generation System™ Kitprovides 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.

Q 02: 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.

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Q 03: Is the insertion site selection of the Entranceposon based on consensus sequence recognition?
Under the optimized reaction conditions of the kit, 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.

Q 04: Is it possible to insert two copies of the Entranceposon in a single target plasmid? 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.

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Q 05: Do I have to use some specific E. coli strain for transformation of the in vitro transposition reaction products?
Make sure that the transformation host strain is sensitive to the antibiotic that you use for selecting the insertion clones. Otherwise, any standard laboratory strain of E. coli that is suitable for high-efficiency DNA cloning can be used, e.g. XL1 Blue DH10B, DH5a.
For direct sequencing of large DNA clones such as BACs and PACs we recommend the use of DH10B as a transformation host.

Q 06: Is any one method more suitable for transformation with the in vitro transposition reaction mixture than the others? Should I use electroporation or can I get enough transformants with the chemical transformation methods?
Any transformation method can be used that is efficient enough to produce more than 106 cfu/µg pUC19 plasmid.
Electroporation is the recommended method for large target DNA clones.

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Q 07: Is there any background problem in the bacteriophage Mu transposition system that is used in the Finnzymes Transposon Products?
No. The Entranceposons that come with the TGS and MGS Kits and also the ones that are available separately are non-replicating linear DNA molecules that are not maintained inside E. coli cells.

With customized Entranceposons that can be constructed using pEntranceposon plasmids some background colonies may appear on the transformation plates. The amount of the background colonies or so called false positives can be reduced by making sure that the restriction enzyme digestion that is used to release the Entranceposon from the vector backbone is complete. It may also be useful to add an enzyme in the digestion mixture that cuts the vector backbone but not the Entranceposon. Sometimes the plasmid minipreps that have been made using the alkaline lysis protocol require an additional treatment with a single-strand specific DNA endonuclease, such as Mung Bean Nuclease, to remove the plasmid form that is not susceptible for the restriction enzyme digestion.

Q 08: 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 insertion?
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 Entranceposonscontain >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.

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Q 09: 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 smear rather than intact band. What are the critical factors affecting the colony PCR reactions?
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 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.

Q 10: Do you have any Entranceposon constructs which would insert an origin of replication and antiobiotic resistance into target DNA e.g. circular bacteriophage genomes so that the target could then be propagated in E. coli?
Entranceposons with various replication origins will be available in the near future.

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