Efficient cloning of linear DNA inserts (ECOLI) into plasmids using site-directed mutagenesis

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This study introduces a novel cost-effective technique for cloning of linear DNA plasmid inserts, aiming to address the associated expenses linked with popular in vitro DNA assembly methods. Specifically, we introduce ECOLI (Efficient Cloning Of Linear Inserts), a method utilizing a PCR product-based site-directed mutagenesis. In comparison to other established in vitro DNA assembly methods, our approach is without the need for costly synthesis or specialized kits for recombination or restriction sites. ECOLI offers a fast, efficient, and economical alternative for cloning inserts up to several hundred nucleotides into plasmid constructs, thus enhancing cloning accessibility and efficiency. This method can enhance molecular biology research, as we briefly demonstrated on the Dishevelled gene from the WNT signaling pathway.

The general scheme of ECOLI—Efficient cloning of linear inserts, as shown on the example of inserting the ECFP tag-encoding sequence into the pDONR221 hDVL3 construct (on the left) and inserting the DEP domain-encoding sequence from hDVL3 into the Flag-mDVL1 (∆DEP) plasmid construct (on the right).

Authors:

Petra Paclíková, Jakub Harnoš

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