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| Molecular Cloning Forum DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here. |
Problems Cloning Large DNA Fragments Together
Molecular Cloning Forum
DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.
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02-07-2008, 09:48 AM
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 Problems Cloning Large DNA Fragments Together Hey!! I try to clone a 5.5 kb DNA fragment into a smaller PGL3 luciferase vector 4.8 kb vector without any success. Despite the 3:1 molar ratios and dephosphating techniques is possible to ligate a fragment of that size into a smaller vector? Is it also possible for the competent cells to take up such a large plasmid after cloning of 11kb plus?? Should i use electrocompetent cells? Also will transfection of mammalian cells with a construct of this size get good transfection efficiencies??  Thanks for any advices        |
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