PCR-after-ligation method for cloning of multiple DNA inserts

PCR-after-ligation method for cloning of multiple DNA inserts
From  Sciencedirect
http://www.sciencedirect.com/sci ... i/S0003269710002198

Outline of the PCR-after-ligation method for efficient multiple DNA insert cloning. The DNA inserts and vector are digested with restriction enzymes to obtain compatible termini, followed by purification and ligation (step 1). Most of the products obtained should be either non-full-length DNA fragments or inverted repeat fragments by self-ligation. Then PCR is performed to amplify ligationproduct using flanking primers, and the DNA fragment with expected size is obtained by gel purification (step 2). The purified DNA fragment is inserted into the linearized vector, followed by transformation into E. coli (step 3).

Agarose gel shows the products obtained from PCR-after-ligation. Lane M: DNA size marker; lanes 1–3: PCRproducts amplified by using the ligationproducts with different molar ratios of vector/inserts as templates. The molar ratios of vector/inserts for lanes 1, 2, and 3 are 1:1:1:1:1, 1:2:3:4:5, and 1:3:1:3:1, respectively.