One-Step Preparation of a TA-cloning Vector from a Specially Designed Parent Plasmid Containing a Dual <Emphasis Type="Italic">lacZ</Emphasis> Gene System |
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Authors: | Soo Youn Jun Seong Jun Yoon Sang Hyeon Kang |
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Institution: | (1) iNtRON Biotechnology, Inc, Room 903, JungAng Induspia V, 138-6, Sangdaewon-dong, Jungwon-gu, Seongnam-si, Gyeonggi-do, 462-120, Korea |
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Abstract: | A high-yield method was developed for producing a TA-cloning vector suitable for blue/white colony selection from a unique
parent plasmid containing a dual lacZ gene system through a one-step restriction enzyme digestion, which creates a single-base 3′-overhang. The dual lacZ gene system was realized by inserting an inner lacZ gene between two single-base 3′-overhangs, creating restriction enzyme sites within the reading-frame-adjusted outer lacZ gene sequence in the parent plasmid. The proposed method overcomes problems, such as the inefficient digestion frequently
observed when generating a TA-cloning vector and the difficulty of purifying TA-cloning vectors from the digestion mixture,
while maintaining the applicability of blue/white colony selection. Moreover, the use of TA-cloning vector prepared by the
proposed method can provide the distinguish tool of transformants carrying the cloning product from those carrying contaminating
parent plasmids, recircularized plasmids derived from incompletely digested parent plasmid fragments, or intra-molecularly
ligated TA-cloning vectors derived from T-overhangs missing TA-cloning vectors (instability of the T-overhangs is another
important consideration when designing TA-cloning vectors) by making all colonies except those carrying the cloning product
appear blue during blue/white colony selection. |
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