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Week_6 - Bi 1x, Spring 2010 Week 6 Session 1 o TOPO cloning...

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Page 1 of 3 Bi 1x, Spring 2010 Week 6 Session 1 o TOPO cloning reaction o Bacterial transformation and plating Background TOPO mediated DNA cloning When sequences are amplified with PCR, the Taq polymerase enzyme adds an extra (untemplated) deoxyadenosine ("A") on the 3'-end of the DNA fragments. These "A-overhangs" are complementary to the deoxythymidine "T" overhangs on the cloning site of the vector. The vector is supplied in the linear form and when you add a PCR product the complementary ends base pair to form a circular plasmid containing both vector and insert. The TOPO name in the kit refers to the fact that the vector contains topoisomerase I that is covalently bound to the vector at the insertion site. The topoisomerase enzyme has a ligase activity that ligates the phosphodiester backbone of the insert to the vector. 1 The vector In addition to 5'- "T-overhangs", the plasmid vector pCR4-TOPO contains the genes encoding for ampicillin and kanamycin resistance. Bacteria transformed with the plasmid can then be
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This document was uploaded on 01/03/2012.

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Week_6 - Bi 1x, Spring 2010 Week 6 Session 1 o TOPO cloning...

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