Help! E.coli transformation - (Feb/22/2007 )

I've been having problems transforming my pet28b plasmid with an insert into E.coli rosetta strain, cause I need tonnes of plasmid. Everything grew fine, except my negative control plates (sterile water+competent cells) grew as well on LB+antibiotic plates. I don't know what's the problem, used a fresh batch of media but it's the same...and I don't know if I should continue selecting for colonies with plasmids or not .... =.="


Thanks in advance

it seems your competent cells were contaminated! prepare new competent cells and go ahead.

perhaps your equipment was also contaminated. Use new tip, new electroporation cuvettes and clean out your pipette, remove the pipette barrel and give it a good long soaking in hot diluted acid. (make sure you wash off the acid after that)

How old are the plates? How old is the antibiotic?

If you have the same amount of colonies in your negative control than in your transformated plates, I don’t thing you should continue picking colonies (could be a waste of time).

I agree with dodosco. I think your competent cells could be contaminates.
If you prepare your own competent cells, check a single colony (o a bit of your –80°C stock cell) in LB without antibiotic and in fresh LB-antib at the same time to see if they are contaminated.

If your think it could be the antibiotic, you can check it plating a clone with a plasmid resistant to other antibiotic. It suppose, that won’t be able to grow up.

Be sure you add the right antibiotic in the right amount to the not very hot LB-medium (50°C aprox).

usually i found my cells and equipments in quite good condition but i suffered 2 times for transformation problem due to antibiotic. i used concentrated antibiotic stock when i was newbie.

usually i found my cells and equipments in quite good condition but i suffered 2 times for transformation problem due to antibiotic. i used concentrated antibiotic stock when i was newbie.

Thanks for all the feedback

Checked the viability of the antibiotics, contamination etc. everything was fine. It seems like the stock prepared by my supervisor contained the pet28b plasmid, same plasmid as what I was trying to transform into the E.coli. I guess that's why my negative controls grew since they had the resistance gene as well

To quote the great philosopher, H. Simpson,


"D'Oh!"

See, even a supervisor can have a bad day!