Advantages of the Gateway cloning method Compatibility and flexibility Since the ccdB product is toxic to the cell, Gateway cloning efficiency can reach >99%.īe sure to verify the integrity of your expression clone via sequencing or restriction digest! Then, you can transform or transfect the cells that you want to use for your experiments and verify that your construct is functional. The reaction between the entry clone and destination vector creates two products: the desires expression clone and a byproduct containing the ccdB gene. #SNAPGENE CLONING HOW TO#This video demonstrates how to use the Snapgene program to design Gateway plasmids.įigure 6: Generating an expression clone. If you choose this strategy, it’s important to include the proper protein expression elements (ribosome recognition sequences, start codon, stop codons, reading frame considerations, etc). In this case, we would use PCR to add att B sites to either end of the KRAS coding sequence. Method A: recombination of an att B- PCR product or plasmid with an att P donor vecto r. There are a few different ways to generate our desired entry clone - human KRAS flanked by attL sites. To better understand the process, we’ll walk through an example experiment where we might use Gateway cloning to generate our desired constructs: lentiviral expression of the human KRAS gene in mammalian cells. Read our recent Plasmids 101 Post on CcdB for more information. coli strains, most colonies should contain the desired, recombined construct. The ccd B gene is present in the donor vectors and the destination vectors prior to recombination, and it is exchanged with the gene of interest during the BP or LR reactions. Since the CcdB protein inhibits the growth of CcdB sensitive E. The entry clone and destination vector carry different antibiotic resistance markers (indicated here by plasmid color), allowing you to easily select for the expression clone. coli cells and select the positive clones. Once the BP and/or LR reactions are performed, the next step is to transform competent E. As in the BP reaction, a DNA fragment containing the ccd B gene is excised from the destination vector. As a result, an expression clone with the DNA of interest flanked by att B sites is generated. This reaction is catalyzed by the LR Clonase enzyme mix. The LR Reaction takes place between the att L sites of the generated entry clone and the att R sites of the destination vector. The LR reaction creates an expression clone with all of the components necessary for gene expression. The BP reaction creates an attL-flanked entry clone. Under certain conditions, the att L and att R sites can recombine, leading to the excision of the phage from the bacterial chromosome and the regeneration of att P and att B sites.įigure 2: The Gateway system adopts phage integration into the BP and LR reactions. As a result of recombination between the attP and attB sites, the phage integrates into the bacterial genome flanked by two new recombination sites ( att L-left- and att R-right-, Figure 1). I n vivo, t hese recombination reactions are facilitated by the recombination of attachment sites from the phage ( att P) and the bacteria ( att B). The Gateway cloning method, developed by Invitrogen, is an in vitro version of the integration and excision recombination reactions that take place when lambda phage infects bacteria. Keep reading to learn more about the Gateway cloning method and its advantages. With the appropriate entry and destination vectors, one can use Gateway to clone a gene of interest into a variety of expression systems. Since its invention in the late 1990s, Gateway cloning technology has become very popular as a rapid and highly efficient way to move DNA sequences into multiple vector systems. Instead, you can choose a molecular cloning technique that will work well with a given set of resources, time, and experimental needs. When facing a cloning project, scientists are no longer limited to traditional restriction enzyme cloning.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |