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Can bacteria grow without a plasmid or am i stupid? - or did ampicillin wear out? (Jun/20/2007 )

I am trying to clone PCR products in to TOPO vector. I am using Invitrogen TOPO-TA cloning kit. its the PCR4-TOPO. The problem is, i ligated the pcr product in to topo vector, transformed (here i used a control plasmid too i.e. puc19) and grow the bacteria. I saw colonies the next day in the pcu19 control (where the number of colonies are more) and the pcr ligated topo plates. The plates have 100ug/ml ampicilin. Then i grow the culture in liquid LB ON at 37 deg in 5 ml and tried to do miniprep (i am using quick plasmid kit from invitrogen) to extract plasmid and ran the gel, i am seeing bands only in the puc19 controls and not in the pcr ligated sample lane. I am also seeing a very faint band in both puc19 control and sample at 12kb...could it be genomic DNA? How could bacteria grow with out plasmid? or am i missing something here? Please help me


Ampicilin degrade with time. Ampicilin also degrades faster when exposed to high temperature. Was the Amp stocks defrosted using high temperature, or perhaps were the plates old?

I wouldn't use plates more then 2 weeks old.


quite true, As ampicillin degrades, this promotes growth of the all bacteria which don't have the resistance gene.


You could check your plates by streaking out untransformed competent E. coli, which should give you no colonies if your plates (and competent cells too) are OK.


QUOTE (perneseblue @ Jun 20 2007, 12:27 PM)
I wouldn't use plates more then 2 weeks old.

i make above 100 amp plates together and store it in 4c refrigerator and use for months

-T. reesei-

I think so Amp