competent cell didn't grow in LB - (Nov/08/2007 )
I'm trying to follow a CaCl2 procedure for preparing competent cells and I'm stuck already on the first step. I streaked my comp cells (TOP10) on a plate, incubated for 20 hours and then picked one colony and put it into 100ml pre-warmed sterile LB in a 1L flask. The next step says to shake at 37 for 3 hours and then check the OD600. Well, I could tell right away when I took the flask out of the shaker...nothing grew 
. How's that even possible? I definitely picked up the colony. Any thoughts?
Hi,
3 hour-incubation seems a bit short to me. Have you shaked them a bit longer (maybe ON) to be sure that they are growing?
Well I'm only new to this type of thing so I don't know how helpful I can be, but did you go ahead and check the OD anyway?? I know when I made competent cells the last time, in the beginning I was worried I was getting slow growth because the media didn't look very cloudy to me at all, but when I measured the OD600 it was close to 0.1
Aside from that, to begin with I would take a colony and inoculate a 10ml culture overnight (I usually do 1 colony in 10ml, then take 1ml of this into a further 9ml for a 10-1 just to be on the safe side).
Then the next morning I take 1ml of each of these into a seperate 100ml of LB each (or 4ml into 400 if I need lots) and then check the OD600 every hour. Having the two different cultures increases your chances of one being successful......
Good luck!!
Yeah, I thought 3 hours seemed short too but this is a protocol that comes from the Molecular Cloning manual book (the one many people call "the bible") and I've found several similar versions online so...
and yes, I actually did check the OD anyway and it was .008 
...not good. So tonight I'm growing a 5 ml innoculum from a new colony and I will innoculate it into 100 ml tomorrow, then it should be up to .3-.5 within a few hours right?
I hope I didn't offend you in asking that, I just wanted to check
3-5 hours sounds about right, my last batch took 4 hours (400ml culture) but I don't know how much this varies with strain. Again, good luck!!!
I hope I didn't offend you in asking that, I just wanted to check
3-5 hours sounds about right, my last batch took 4 hours (400ml culture) but I don't know how much this varies with strain. Again, good luck!!!
Oh, no not at all - the eye-roll was for my own annoyance at my incompetence! I actually almost threw it away because it looked so clear and it was so obvious that nothing had grown. But something in my gut told me...just measure it anyway...it can't hurt. So much for my gut.
Anyway, my inoculum grew last night so now I'm waiting on the bigger batch to reach the right OD. Thanks for the luck wishes! So it's been about 6 hours since I innoculated that flask and it's at about .25 OD600. It's supposed to take 2-2.5 hrs to get to .35. Why is this happening?? I've done everything exactly according to the directions. Somebody kill me!!
Damsweet, you haven't grown an overnight culture before you inoculate your 100 mL of LB for 3 hours. You can't just grow up a thick culture of cells in 3 hours from a single colony, think about how long it usually takes to grow up a culture. Take your single colony and grow it in 10 mL of culture overnight, then take 1 mL or whatever of that culture in the morning and inoculate it into 100 mL of LB and grow until OD600.
Secrets to good competent cells:
-stop the cell growth just before OD600 - to allow for the small amount of growth after you take the culture out of the incubator
-stop the growth immediately - you want to stop them growing there because that's when they're in the log phase and multiplying - i.e. where they are most competent/most likely to recover from the shock of transformation
-keep them as cold as possible for the entire procedure - that keeps them in the log phase
Good luck,
Rob
Good luck,
Rob
Read it again..that's actually exactly what I did (the second time). Grew a 5ml innoculum overnight and transfered 1ml of that into 100ml LB. But after 7 hours, when I decided to just go home last night, it still hadn't reached .3 @600 - which should have taken 2-2.5 hours according to all protocols that I've read. That's exactly the problem - I follow instructions to the T and still...
Check the pH of your culture medium and the temperature of your incubator. Either could cause slower than normal growth. If you have antibiotic selection, some antibiotics (Tet especially) can cause slower growth even in resistant strains.