questions about transformation
Posted 21 February 2011 - 12:26 AM
Afterwards, i grow the bacteria on LB plate with kanamycin. I then pick the colonies and grow it in LB medium with kanamycin
But then those bacteria cannot grow in the medium, i wanna ask why?
Some of the medium with bacterial grwoth, i then isolate the plasmid using plasmid purification kit with silica-gel membrane. Why the yield is not high?
Thanks so much
Posted 21 February 2011 - 12:50 AM
Posted 21 February 2011 - 02:23 AM
Posted 21 February 2011 - 03:34 AM
Posted 07 March 2011 - 01:02 AM
1. The donor plasmid which contains the KanR was carried over after the gel extraction, if it's the low copy number plasmid, this could explain for the case that you get the low yield of pladmid. It happened to me many times, even though I excise out the low band, some of the intact plasmid still contaminated.
2. You've got the contamination, they can resist to the Kan on the plate but they will die if you grow them in the medium.
I suggest to spread your transformed cells on the Kan + Amp plate for the accurate screening.
If then you still have the low yield of plasmid, you can do the control experiment by prep the original pUC, it's the high copy number plasmid. In the case you get the low yield, you should check your kit, it might be in trouble.
Some point mutations can reduce the copy number so you get the low yield, but if it happens every time, mutation can't be the reason.