Colonies from transformation plate are not growing...:(
Posted 21 July 2012 - 05:44 PM
Posted 21 July 2012 - 11:53 PM
It could be that the plasmids are toxic to the bacteria.
Posted 22 July 2012 - 01:43 AM
I would not use BL21(DE3) for cloning ...rather use a cloning strain that does not express your gene of interest and then subclone your plasmid into your expression strain once you have confirmed your insert.
Maybe your protein is toxic ...and they are able to grow once on solid medium ...but then get rid of the plasmid rapidly ...and the cells you use for inoculation are already dead or do no longer contain the plasmid.
What kind of gene you tried to clone and express?
Posted 27 July 2012 - 03:46 AM
Posted 12 August 2012 - 05:31 PM
Additionally, another step of confirmation would be to control digestion with your restriction enzymes you used for digestion. Did you try liquid culture?
Last, but not least, I would agree also with pDNA; do not use BL21DE3 for your cloning. In case that your protein is toxic you could use specific strains whose genes responsible for toxicity have been deleted. Strain like these is C41 or C43.
I hope that helps.
Posted 13 August 2012 - 03:43 AM
What is this LB-Kanamycin 70? Are you using 70 ug/mL kanamycin? The recommended concentration is 30-50 ug/mL final concentration. 70 would a tad too much.