Cloning strategies, Part 2: Cohesive-end cloning

Cohesive-end cloning is one of the most commonly employed techniques in molecular biology. Review these tips and tricks for cloning using restriction enzymes.

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Isothermal assembly: Quick, easy gene construction

Learn how, in a single reaction, isothermal assembly can combine several overlapping DNA fragments to produce a ligated plasmid ready for transformation.

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Methods for site-directed mutagenesis

Review these traditional PCR-based methods for creating a specific mutation in a known sequence, in vitro. Then read our follow-up article, Site-directed mutagenesis—Improvements to established methods (see the "Additional reading" sidebar) which describes how you can generate the same types of mutations, more quickly and efficiently, using custom, synthetic dsDNA fragments.

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Increase oligo stability with phosphorothioate modifications

Modification Highlight: Need oligos that are nuclease resistant? Phosphorothioate bonds substitute a sulfur atom for one of the non-bridging oxygen atoms in the phosphate backbone of an oligonucleotide. Resistant to both endo- and exonucleases, this linkage provides increased oligo stability.

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Starting with RNA—one‑step or two‑step RT‑qPCR?

Starting with RNA? When performing real-time qPCR, one has to decide whether to use a one-step protocol that combines the RT reaction and PCR in one tube, or a two-step protocol where the RT reaction is performed separately from the PCR. Here are some guidelines.

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