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pYES2.1 TOPO TA Expression Kit From Invitrogen
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Full Product Review
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The pYES2.1 TOPO TA Expression Kit allows you to clone a gene via PCR without using the often frustrating ligase enzyme. You can produce a PCR product, ligate into the pYES2.1 /V5-His-TOPO vector and transform the new recombinant vector into E. coli in the same day. The kit takes advantage of the fact that all PCR products amplified with Taq polymerase leave a single deoxyadenosine (A) to the 3’ end of the PCR product. The TOPO cloning vector, which is supplied in the kit, has a single complimentary overhang, a 3’ deoxythymidine (T) residue, allowing a ligation between the two products. Topoisomerase is attached to the vector, allowing the vector to be “activated”, thus providing enough energy to spontaneously drive the reaction. The reaction takes only 5 minutes, although we always allowed the reaction to incubate for 30 minutes to achieve maximum efficiency.
Once the ligation reaction has taken place, the product can be transformed into E. coli cells. The kit is supplied with chemically competent TOP10F’ One Shot cells and growth medium (SOC media), which can be transformed in under 2 hours. You will need to have a 37ºC incubator and ampicillin plates to select for transformed colonies. After an overnight incubation, you must screen for transformed colonies via restriction analysis or sequencing; there is no blue-white screening for this vector. Once you have successfully identified a clone with your insert, you can transform it into yeast and use galactose to induce expression of your cloned gene. We have no experience doing so, as we cloned our gene with its endogenous promoter, which worked fine to express our gene. |
Review Synopsis
Product: pYES2.1 TOPO TA Expression Kit From Invitrogen
The Good: Quick one-step cloning; can be used for direct insertion of Taq polymerase-amplified PCR product with no need to ligate; product can be transformed into yeast; protein can be expressed with galactose promoter and purified via the V5/His Tag.
The Bad:The kit is expensive. No blue/white clonal selection of transformants. Must restriction digest or sequence to check cloning of gene
The Bottom Line: This is a great kit to use for someone who wants to clone a gene into yeast without the hassle of a ligation. It works well and saves you valuable time. Having an inducible promoter is a plus for studying the protein in vivo. |
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The pYES2.1 TOPO TA Expression Kit includes the pYES2.1/V5-His TOPO vector, all the buffers and solution for the “ligation” reaction, control primers and plasmids, TOP 10F’ cells, SOC media, and control sequencing primers. The kit does NOT include the polymerase enzyme for PCR.
Although this kit is definitely expensive, we found it worked well for expression of a gene that we had trouble cloning by the traditional “ligation” method. It is quick and saves a lot of time, so in the end, it may be worth the money you invest for all the time you save. A great advantage for a yeast geneticist is that this shuttle vector can be introduced into yeast, allowing for protein expression and function studies in vivo.
Author
Haley Curtis Stevens
Graduate Student
Department of Physiology
Emory University
United States |
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