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Fig. 3 | BMC Biotechnology

Fig. 3

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

Fig. 3

Deletion and replacement of residues within hChR2. a Depiction of the hChR2-mCherry synthetic gene (colored segments, A and B). Fragment A includes sequences spanning from the standard promoter of the plasmid and intracellular linker III (ILIII; purple); fragment B spans from ILIII to the end of the mCherry (orange). Backbone of plasmid is light grey. The mutations (cyan) are situated between the purple and orange fragments. Sizes (# of bps) of the fragments are noted within the fragments. Intrinsic and unique digestion sites are indicated (BamHI and BsrGI). Primers used for amplification of each fragment are noted on the right (with corresponding colors). b Image of PCR products from step I on 1% agarose gel. DNA ladder sizes (in Kbp) are noted on the right of ladder. c The amplified assembled Gibson products, obtained from varying incubation times (15 to 120 min) by CAG promoter_F and the FP_R primers (left), are visualized on 1% agarose gel and analyzed by enzymatic digestion (right panel, BamHI and BsrGI). d Sequences, and matching chromatograms, of DNA sequences obtained from resulting colonies (dashed box)

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