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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. |
No Positive Colonies or Clones!
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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04-18-2008, 02:33 PM
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 No Positive Colonies or Clones! Hello I have been attempting to transform E.coli DH5 alpha cells with my insert (1.4kb insert into vector 7 kb). The insert has been well digested with HINDIII and blunt ended for the other end. For the vector I ligated O/N at 16C using 1ul vector+2ul insert+3ul enzyme. I grew the cells in LB medium containing Kan and Hyg (50/ul). However I have no colonies! Any ideas where I went wrong? thanx        |
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04-18-2008, 02:43 PM
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 Re: No Positive Colonies or Clones! 1) Are your competent cells competent? Do you have a positive transformation control? 2) Do you have an estimate how much DNA you added to the ligation -- in terms of nanograms and/or molarity? What kind of vector:insert molar ratios did you try? 3) What kind of competent cells did you use? Chemically competent? Electrocompetent? Did you let the cells recover from transformation before adding antibiotic? |
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| cloning, dna, insert, plasmid |
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