Zymo DNA/RNA shield protects DNA from freeze thaw cycles, stabilizes nucleic acids and deactivates nucleases. In order to facilitate the Golden Gate Assembly, fragments to enter the assembly, previously amplified by PCR can be stored in Zymo DNA/RNA Shield at a ratio of 1:3 (PCR : Zymo Shield) thus eliminating the need to store the fragments in plasmids and facilitating the screening of the desired mutants. The modular vector designed in this paper contains 6 fragments or modules that can be easily interchangeable. Furthermore, these types of vectors can replicate during cell division in certain types of cells. It also provides the vector with the ability to remain episomal and be mitotically stable during cell division. It contains a Scaffold/Matrix Attachment Region which is an element designed to permit the persistence of the vector as an extrachromosomal element within a host cell. A S/MARs vector is a type of non-integrating episomal vector that is used for mammalian expression. In turn, herein described is a simple inexpensive protocol for standardizing and assembling several fragments with good fidelity, in just one day with minimal hands-on time.įor this purpose a modular S/MARs mammalian expression vector (called pNoname) has been designed. However, although standardized and agreed by the community, these standards may prove too complex for assembling just a few/several fragments or for students that are just getting started in molecular biology or that have a desire for more customization. The level 1 transcriptional units can be, in turn, ligated into level 2 multigene constructs using a different type IIs restriction enzyme. Level 0, or basic modules (Promoters, 5’UTR’s, Signal Peptides, CDC’s, 3’UTR’s and Terminator regions) are assembled into Level 1 transcriptional units using a Type IIs restriction enzyme. Mo-clo or similar standards like Golden Braid 2.0 use 3 levels of successive assemblies. Mo-Clo consists of standard parts, or basic modules, of eukaryotic Promoters, 5’UTR’s, Signal Peptides, CDS’s, 3’UTR’s and Terminator regions, each module containing a standardized 4 nucleotide overhang. Mo-Clo, or the modular cloning standard, is one of these standards. Since the sequence of the overhangs at the ends of the digested fragments can be chosen to be any 4-nucleotide sequence, multiple compatible DNA fragments can be assembled in a defined linear order in a single restriction-ligation step.īased on this method multiple standards to construct multi-gene circuits have been developed. Since the ligated product of interest does not contain the original type IIS recognition site, it will not be subject to redigestion in a restriction-ligation reaction.Īll products that reconstitute the original site will be re-digested, allowing their components to be made available for further ligation, leading to formation of an increasing amount of the desired product with increasing time and cycles of incubation. The principle of Golden Gate Cloning is based on the ability of type IIS enzymes to cleave outside of their recognition site, allowing two DNA fragments flanked by compatible restriction sites to be digested and ligated seamlessly.
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