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Is it the transformation or the plasmid?

Is it the transformation or the plasmid? - Molecular Cloning Forum

Is it the transformation or the plasmid? - 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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Old 02-22-2011, 05:26 AM
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Unhappy Is it the transformation or the plasmid?



I've been trying to transform E.coli cells (JM109, XL-1 BLue and DH5a) with a pET15b vector. The results have been sketchy - only got a single colony of XL-1 Blues on ampicillin plates. The cells grew fine in liquid medium with amp and a miniprep yielded on okay amount of plasmid. However, the company I've sent it to for sequencing cannot find anything: their pcr isn't picking up anything and nothing is seen on a gel. WTH?

Is it possible that the transformation was unsuccessful - and if so, what came out in the miniprep?

Or, the plasmid has been slightly modified but has been successfully used by another group, so I think its unlikely to be that.

Finally - do you think the problem could be with the sequencing??
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Old 02-22-2011, 12:40 PM
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Default Re: Is it the transformation or the plasmid?

When you say you got a decent amount of plasmid but nothing on a gel, how did you test the success of your miniprep?
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Old 06-18-2012, 06:47 AM
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Default Re: Is it the transformation or the plasmid?

I also want to know
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Old 03-08-2013, 08:13 PM
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Default Re: Is it the transformation or the plasmid?

So no one knows the answer to this? I have been trying to clone into a pet15b vector as well (Nde1 and BamH1 sites) and it just won't go in. Does anyone know what's going on?
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pet15b , plasmid , transformation


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