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Plasmid isolation - (Jul/05/2006 )

I am trying to clone GFP 721bps insert into the pTrc His A vector (4.8Kb), this vector already has a part of a gene about 400bps long. So basically I am doing a domain cloning. I have dephosphorylated my vector to ligate the GFP insert into it. And after Transformation, I checked for the insert by Colony PCR, I could get a band of expected size (721bps) on the gel. But my problem is that when I grow the bacteria to isolate the plasmid, I could not get any plasmid after miniprep. I have also tried to isolate the plasmid by chemical method but then too no plasmid. Is it possible that the plasmid has become a low copy number plasmid after ligation. Can anybody suggest me anythig regarding this?



What cells did you use? Some of them ( i think they are often recA plus) have a habit of chewing up plasmids.


Are you sure your minipreps are working well?, did you have a control, may be one or your solutions is not working well.
Did you run on agarosa gel an aliquot of your ligation to “see” the ligation and or use a ligation contol to be sure you have ligation products. The control of transformation is important too (tansformation with a known amount of plasmid) to see if your cells are ok. A negative control too, so be sure your bacteria are not contaminated.
Ones, I was transforming with a ligation product and I always obtained some colonies. I did minipreps without result!!
The problem was that the ligasa was not working, and the bacterias was some how contaminated with a bacteria resistant to amp but without plasmid. I realize the problem because of my negative transformation control and my ligation control.
Some time after that, a friend from other lab has a similar problem, she had some yeast contamination in her bacteria, and they look like bacteria.

-aztecan princess-