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joyce

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    The product of this reaction was then digested with the restriction enzymes EcoRI and PstI. The three vectors chosen to clone EGF were: pET21b(+), pET22b(+) and pET32b(+).When ligating EGF DNA with these three vectors, a HindIII diane was needed, so EGF was first cloned in the pUC118 vector. From this vector it was possible to obtain EGF gene with a simple PCR reaction using the universal primers, FUP (Forward Universal Primer) and RUP (Reverse Universal Primer), and then digesting the product with the restriction enzymes EcoRI and HindIII. With this product and with the three plasmids also digested, a ligation was done. The different transformants obtained were then assessed to see if they were positive for EGF by carrying out digestion with restriction enzymes which would result in fragments of different lengths depending on whether the clone was positive for EGF or not (Fig. 1). Positive clones for each construction were obtained.

    Fig. 1. Verification gel of transformants obtained after ligation between EGF gene and the three pET vectors. Samples were loaded on a 1.5% agarose gel. The different lanes are the digestions with ApaI and BglII of pET21b(1) (lanes 1–4), pET22b(+) (lanes 5–8) and pET32b(+) (lanes 9–12). The markers are also shown (1-kb Plus DNA Ladder). Positive clones are marked with an arrow.

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    2 個解答生物學1 0 年前