I transformed with a ligation reaction into Ecoli comp cells, and let them outgrow in a couple mLs of LB overnight on accident.
Can I plate the transformations anyway and hope that I have colonies with ligated plasmid? Or should I just toss the culture and do it over?
Hi Kara,
If I have understood well. You have done your ligation. And you have let the adaptation incubation (e.g. 250 rpm shaking, 37 °C) ON insted of 30 min-1 h. Am I right? And your question is can you streak your LB-agar plate with this transformation mixture? I think the above mentioned adaptation incubation is good for bacteria after heat shock. But the nutrients (protein, carbohydrates) will decrease in parelell the culture is growing. (saturation curve) Especially in few mLs of LB. So the problem with the outgrown culture is the excess of the dead bacteria (without any transformed plasmid). Maybe you can do the plating. But I think it's totally unnecessary. If you have got colonies, they might be false positives, mainly. If you have ligation mixture left, I suggest to do a fresh transformation, with a max. 30 min-1 hour adapt. inc. after the heat shock.
Bests,
Tamas
Well if your cloned plasmid has an selection marker, you could try to plate it on the chosen media and hopefully from the surviving bacteria, only the ones that have your clone should survive. Worth giving a shot!
If you outgrowth your cloned in just LB or LB + selection marker, however you should verify again by plating on agar plate + selection marker (antibiotic).
If they growth well, you can pick up several colonies with sterile toothpick and verify by colony PCR.
You can use the same primer that amplify your gene target for colony PCR.
Regards.
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