No bacterial bands in uninduced - (May/24/2006 )
I am following Invitrogen's protocol for cloning using pET 151-D TOPO. After growing an overnight culture of BL21 cells, I add 500 ul of Overnight to 10 ml of fresh LB-amp. This grows for 2 hours until the OD is between 0.5-0.8 All is well. I split the culture into two smaller cultures. I add PTG to 1 mM (5ml culture so 5 ul of IPTG) to one of the cultures. I have 2 (5ml) cultures one induced one uninduced. I then take 50 ul of each culture every hour. HERE is the rub:
After processing samples by removing supernatant and boiling in samplebuffer, The uninduced has pretty bacterial bands. The induced only has one dark band all the way across it, and no other bands. Why am I not getting bacterial bands in these samples as well? Any help would be greatly appeciated.
Nail
After processing samples by removing supernatant and boiling in samplebuffer, The uninduced has pretty bacterial bands. The induced only has one dark band all the way across it, and no other bands. Why am I not getting bacterial bands in these samples as well? Any help would be greatly appeciated.
Nail
could it be that your protein was hugely overexpressed, and it is toxic for the bacteria. so you have less bacteria in induced than in non induced sample.
that's why you do not see the bacteria bands, even if you see the huge overexpressed protein?
Did you centrifuge the bacteria after inductin? could you compare the pellets? Did you see a difference in the OD of the medium ? (even just with your eyes)?
Thank you,
I did see a difference in the opacity of the media as the cultures were growing. Uninduced and induced looked the same. Both got cloudy-er with time. Both produced pellets that got bigger with time. There was no discernable difference between induced and uninduced as far as that goes. I will take OD next time.
i agree with Missele, i think you have tooooo much of your protein 
...reduce the loading volume and see what happens.....or dilute in sample buffer before loading....