two fragment ligation - (Feb/21/2007 )

I ligate my gene and vector cut by EcoRI and NdeI;
ligate cut vector
as negative control. Then I transformed these two ligation and
uncut vector as positive control into E.coli. After overnight growth, I got colonies from all plates including negative control. I did PCR by using M13 primers to check these colonies and only got fragment about 1kb in positive control (uncut vector). I think if these colones from negative control contain vector uncompletly cut, these colonies can grow in LB+AMP plate. However, when I did PCR and I didn't get any PCR product . when I culture some colonies from negative control and my gene plate in LB+AMP medium, and extrat plasmid DNA but I got plasmid DNA only from colony of uncut vector (positive control). what are other colonies? How to explain this result? Can anyone help me? Thank you very much

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contamination? I have heard of it happening, does your vector have 2 antibiotic resistance genes or just one?
Is your antibiotic fresh? How big are the colonies?

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my vector has 1 antibiotic gene. I am sure this antibiotic is fresh. some colonies are big and other are small. it is hard to select colonies from plate. I don't think it is contaminated. I did twice. it still like this. I feel it is so strange that I don't get plasmid from colonies growing in LB containing AMP but colonies from positive control plate.

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Smilesmiles,

I had this problem before but was due to my plates. I later realised that the antibiotic was not working. I tried preparing everything fresh and i solved the problem. Also, check your cells to make sure they are not contaminated. Some times it happens that way.

good luck!

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