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Fig. 2 | Plant Methods

Fig. 2

From: Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes

Fig. 2

Determination of Pyrite cloning efficiency using colony PCR and blue-white screen. a Examples of colony PCR from three different reactions (i, ii, and iii), showing about 55% colonies (10/18; star) with the expected insert. “m” indicates marker lane (Goldbio 100 bp DNA ladder). Reaction i screened for insertion of F. vesca gene30478 CDS into pB42AD, reaction ii screened for insertion of F. vesca gene30478 CDS into pLexA, and reaction iii screened for insertion of F. vesca gene25060 CDS into pB42AD. bg Blue-white colony screening used to determine the efficiency of the multiple applications of Pyrite cloning. Colonies are formed on antibiotic containing LB agar plates spread with X-gal and IPTG. b Control experiment showing 100% blue colonies after transformation of vector pUC19. c pUC19 was put into the Pyrite reaction to determine background re-ligation in the absence of DNA fragments. A smaller number of blue colonies was observed. d Pyrite reaction to insert F. vesca gene25060 CDS into pUC19. 100% white colonies were observed. e Pyrite cloning of gene25060 into pUC19 with SalI and BamHI-HF, a heat-resistant restriction enzyme, yielded 83% white colonies. f Simultaneous cloning of a F. vesca gene31413 into two vectors, pUC19 and pSanFran, in the same tube. The result of cloning gene31413 into pUC19 is shown. g Swapping eGFP from pSanFran to pUC19 with the Pyrite reaction, yielded 49.3% white colonies

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