We constructed a methanol assimilation pathway to produce poly-3-hydroxybutyrate (P3HB) using the MCC pathway, which included the ribulose monophosphate (RuMP) pathway for methanol assimilation and non-oxidative glycolysis (NOG) for acetyl-CoA (precursor for PHB synthesis) production. NOT FOR USE IN DIAGNOSTIC PROCEDURES (EXCEPT AS SPECIFICALLY NOTED).The naturally occurring one-carbon assimilation pathways for the production of acetyl-CoA and its derivatives often have low product yields because of carbon loss as CO 2. Our mission is to develop high-quality innovative tools and services to accelerate discovery.įOR RESEARCH USE ONLY. As a member of the Takara Bio Group, Takara Bio USA is part of a company that holds a leadership position in the global market and is committed to improving the human condition through biotechnology. provides kits, reagents, instruments, and services that help researchers explore questions about gene discovery, regulation, and function. Looking for more information about designing projects that use In-Fusion technology? Check out our In-Fusion primer design FAQs for details on specific cloning applications. We highly recommend our In-Fusion primer design tool for step-by-step guidance with In-Fusion Cloning, as it can help you design primers for single- or multiple-fragment cloning, or even site-directed mutagenesis. Still feel unsure? Online tools are available that can simplify primer design and reassure you that you are designing primers properly. We recommend CloneAmp HiFi PCR Premix or PrimeSTAR Max DNA Polymerase. DO use a high-fidelity DNA polymerase to generate your insert to ensure sequence accuracy. Do NOT use low-fidelity polymerases, such as Taq, which can introduce errors into your sequence.Always use PCR-grade water (both as a reaction component and to reconstitute your primers), fresh DNA polymerase and dNTPs, and high-quality DNA template. Before you start, ensure that you have the right materials. Regardless of your specific primer designs, be sure to give your seamless cloning the highest probability of success by remembering these standard best practices for PCR:.In most cases, desalted oligonucleotide primers work just fine. If the quality of your primer is poor (e.g., has many premature termination products), or is longer than 45 nucleotides, a PAGE-purification step may be necessary. Primer quality depends on the vendor and can vary from lot to lot.LALIGN is another free sequence alignment tool that can help you identify potential unintended targets within your template sequence.You can perform a BLAST search to see if the 3′ portion of your primer might hybridize to unintended sites on your template.Complementarity between primer pairs to avoid primer dimers.Complementarity within your primers to prevent hairpin structures.The last five nucleotides at the 3′ end should contain no more than two guanines or cytosines. Runs of identical nucleotides in the 3′ portion of the primer.All remaining cycles should be based on the full-primer T m. NOTE: During PCR amplification, the first-cycle T m should be based on just this 3′ portion of the primer. If the T m is too low, increase the length of the gene-specific portion to reach a T m between 58–65☌. The primer T m is calculated from the 3′ (gene-specific) end of the primer, NOT the entire primer.(See Tip 4 for details on calculating the T m.) If the difference between the T ms for your forward and reverse primers is >4☌, you are not as likely to get efficient amplification. Aim for melting temperatures (T ms) between 58–65☌.If you are using In-Fusion Cloning, this overlap can be shortened to 15 bases for single-insert cloning and 20 bases for multiple-insert cloning. Most seamless cloning protocols, including the Gibson method, require the 5′ end of the primer to contain 25 bases that are homologous to one end of the DNA fragment to which it will be joined (i.e., the vector or another insert).This sequence should be 18–25 bases long and should ideally have a GC content between 40–60%. The 3′ end of the primer should contain sequence that is specific to the target gene you are amplifying.Interested in trying seamless PCR cloning but don't know where to start? Or maybe you just need a quick refresher? Here's a list of top tips to keep in mind when designing your primers for seamless cloning, including some information specific to In-Fusion Cloning.
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