Fig. 3

Multiple applications of Pyrite cloning. a Circularization/ligation of a linear DNA fragment. A PCR amplified linear vector can be cut and ligated using the Pyrite reaction. pLacZi was amplified with primers MF143 and MF144 and put in the Pyrite reaction with HindIII-HF, restoring the HindIII site in pLacZi. MF41 and MF126 are PCR primers used to test successful cloning. Three of the eight colonies tested were positive and marked by white stars. b Insertion of a DNA fragment into multiple destination vectors in a single reaction. The transformed cells can be plated on different antibiotic containing media to select for the desired vector. The gel image on the left shows the insertion of eGFP into two vectors simultaneously; the image on the right shows the insertion of gene31413 CDS into two vectors simultaneously. Red star indicates negative control (no template). Green stars indicate the vector pMerlin, which confers AmpR. Orange stars indicate the vector pSanFran, which confers SmR. “m” indicates marker lane (Goldbio 100 bp DNA ladder). c Restriction digestion of extracted plasmid DNA by EcoRI and SalI to release the cloned fragment. Proper insert and vector size confirmed colony PCR results. Arrows indicates the released insert of 817 bp and 727 bp respectively. Green star indicates vector that conferred AmpR (pMerlin). Orange star indicates vector that conferred SpecR (pSanfran). m indicates Goldbio 1 kb DNA ladder. d Insert swapping between vectors with different antibiotic resistances. Two successful examples are shown with 67–100% efficiency (positive PCR products marked by white stars). e A vector with an insert can revert to the empty vector by releasing the insert with the Pyrite reaction. Gene31413 CDS was removed from the pCR™8/GW/TOPO^® vector with EcoRI-HF. All colonies screened contain the empty vector without an insert (blue stars)