Template Generation System II Kit - FAQs

View additional product information for Template Generation System II Kit - FAQs (F702)

6 product FAQs found

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.

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.