![]() While cloning efficiency was low (between 6 and 20%), high colony numbers made it possible to identify a colony containing the full-length insert in both cases. Sixteen colonies were analyzed to calculate cloning efficiency ( Figure 3C). A 10 μL aliquot was then used to transform ElectroMAX DH10B electrocompetent cells. To maximize transformation efficiency, the assembly product was precipitated with 100% ethanol and resuspended in nuclease-free water. Two and four fragments were assembled into the pASE101 vector to build 25 kb and 50 kb inserts, respectively ( Figure 3A and 3B). The inserts and vector with 50 bp of homology at each end were amplified using Platinum SuperFi II DNA Polymerase and purified with a GeneJET Gel Extraction Kit. In Figure 3, the GeneArt Gibson Assembly EX Cloning Kit was used to build large DNA constructs in the low-copy pASE101 vector (2). Regardless of your choice, GeneArt Gibson Assembly HiFi and EX Cloning kits are available with both chemically and electrocompetent cells. ![]() While either method is suitable, the use of electroporation with electrocompetent cells often provides higher transformation efficiencies, but the process will require an electroporator.Īs such, your method of selection will primarily depend on your lab setup. In this case, simply cut the insert using restriction enzymes, purify, and then add to the assembly reaction with your vector and stitching oligonucleotides.ĭuring transformation steps, bacterial host cells can be prepared to take up foreign DNA using of two methods: electroporation or chemical transformation. This strategy is helpful when working with large (>10 kb) or complex DNA inserts with high GC content or secondary structures. The technique can be used in several ways, including carrying out a cloning strategy without generating a new PCR amplicon, or cloning the same PCR amplicon in multiple vectors without repeating PCR amplification of the fragment. And your downstream uses for it dictate how much you need, what contaminants you can tolerate, and your extraction and purification methods. This approach offers flexibility and enables virtually any possible combination between DNA fragments ( Figure 2). Most experiments start with a piece of DNAeither plasmid DNA or genomic DNA. When doing one- and two-fragment cloning, two or three stitching oligonucleotides can be used with the Gibson Assembly method. Stitching oligonucleotides work as a bridge between the DNA fragments to be joined by sharing half of the sequence with each fragment. How can you use Gibson Assembly when your DNA fragments do not share any homology? The answer is to use stitching oligonucleotides.
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