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Fig. 1 | BMC Biotechnology

Fig. 1

From: A simplified Gibson assembly method for site directed mutagenesis by re-use of standard, and entirely complementary, mutagenesis primers

Fig. 1

The modified Gibson assembly method using SDM primers and added quality control steps. Flowchart of the method’s main steps. Step I—PCR amplification of the DNA sequence by use of standard SDM primers (50 bp; purple–orange arrows) containing the desired change in sequence, i.e., mutation (green highlight). Each SDM primer (sense, + ; antisense, −) is used separately to amplify the fragments (A and B) flanking the site of mutagenesis (green). This is achieved by the additional use of regular amplification primers (~ 20 bp, short purple and short orange arrows). Step II—A and B amplicons are assembled using the Gibson reaction mixture. Step III—the resulting assembly is amplified by the same primers employed in step I. Note that these primers can only amplify the assembled (correct) fragments. The large amounts of product obtained by this amplification can be used to (1) visualize and examine size of products by electrophoresis (1% agarose gel, cartoon) and (2) products can be isolated and sent to sequencing (cartoon chromatogram). These are the added quality control steps (QC) introduced. Step IV—The amplified assembly product is digested and ligated into a desired plasmid (vector) and transformed into competent cells. The entire process spans 4 days

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