I was doing transformation using plasmid with kanamycin resistance into BL21 DE3 E. coli cells with ampicillin resistance. I plated the culture on amp+kana plates and amp only plates. However, only colonies were found on the amp only plate. Since the transformation has fail several times before this, I decided to inoculate the colony from the amp only plate and continue the experiment (grow the culture to 0.55 OD600 and add IPTG). Afterwhich I ran a SDS-PAGE. I observed a difference between before IPTG and after, and the protein bands in the after IPTG lane corresponded to my protein of interest, which should not have been expressed... Does anyone have any knowledge about this? Please help.
Did you do a western blot to confirm? Many metabolic proteins are overexpresed after iptg induction, also you need to do a pcr to confirm the plasmid transformation?
What is the concentration of kanamycin in the plates? We use 30 - 50 ug/ml.
In the transfornation, after plating, i add more media and antibiotics to the remaining liquid culture, and let it shaking overnight, if there are no colonies, i plate the ON culture.
Hi Jahaziel Gasperin Bulbarela
Thank you for your advice and suggestions, I have not done a western blot and PCR, maybe I'll give that a try first.
And I used 50 ug/ml of kanamycin in the plates.
Do you also know if using pure agarose vs bacto agar will affect the growth of the colonies after transformation?
What is the source of the Ampicillin resistance?
But there are going to be a few different proteins expressed with IPTG induction one of which will be the T7 polymerase that may well have many metabolic consequences.
If your gene is only on the Kan plasmid then it likely is not really your protein being expressed but you can confirm by western blot as suggested by Jahaziel Gasperin Bulbarela
There should not be any difference in using agarose vs bacto agar (other than a large difference in price).
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