Dear colleagues,
I transformed DH5alpha with 70 ng or pUC-based construct per protocol (C2987 NEB). There were not many colonies seen next day (approximately 20 tiny colonies after 1:10 dilution with SOC) as opposed to hundreds good-sized colonies with various constructs, including this one during other transformaitons). I picked two colonies and started an overnight 16-hr incubation in LB at 37C for 200 rpm for a miniprep. The opacity was quite low and the streak plates showed extremily tiny colonies after an overnight incubaiton.
What could be the cause for this?
I recall thawing these bacteria for too long (about 20-30 min) on ice, more than 10 minutes(1st step of transformation) The other steps of transformation were not changed. Could it be a reason for poor bacterial growth now?
Kind regards,
Maria
I doubt that thawing too long was the issue. Mishandling the competent cells might have an impact on how many colonies you get, but once they start growing then the "history" will have gone away and the colonies should behave normally after that. So it sounds like it may have more to do with this particular plasmid.
@Michael Benedik, thank you for the reply. it seems like the construct is toxic to cells.
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