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competent cell didn't grow in LB - (Nov/08/2007 )

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QUOTE (Damsweet @ Nov 11 2007, 02:56 AM)
QUOTE (killerkoz17 @ Nov 10 2007, 12:00 AM)
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.

Good luck,

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... mellow.gif

Ah yep, fair enough (it was hidden in the post). Well if the overnight culture is growing well that suggests the cells and the media are ok. Maybe you are growing the cells for too long overnight(?), so they are passing the log phase (growth) and entering the stationary phase (cell growth = cell death)? I'm not sure how likely that is or if it will affect your morning culture but it may be a possibility. I'm really not sure but I'll keep thinking about it.


hmm...well we buy our LB (since we don't use it much) so I'm assuming it comes in the right pH? Maybe I'll check it anyway. Since I'm at my wits end. I streaked out electrocompetent cells on Friday and the picked colonies from both the electo and chem. compet. cell plates and made two more 5ml innoculums. I grew them overnight in snap caps at 37C shaking at 300RPM for 20 hours (is that too long?), they grew fine so this morning I took 1 ml of each and put into 100 ml fresh LB. Came back 3 hours later - OD600 is still under .1 blink.gif blink.gif blink.gif blink.gif wacko.gif


I think killerkoz17 has a good point about the bacteria growing too long and reaching stationary phase, I really think its worth a shot to do dilutions of your culture (I think I mentioned this in one of my last posts, so if you have tried it already- sorry!!) eg.

1. take a single colony into 10ml LB, mix well
2. take 1ml of this into a further 9ml (I guess you could do further dilutions as well, if you wanted to be thorough.)

Then the next day make a larger culture from each of your smaller ones and hopefully one will make it!!!

Once I was using a bacterial strain that was a being a little difficult, and in the end I had to make up 4 different cultures just to get one to work. Its a lot of work to begin with, but if one works- its so worth it!!!


yeah so...don't ever use a nanodrop to OD your samples............ closedeyes.gif ph34r.gif ph34r.gif ph34r.gif ph34r.gif


QUOTE (Damsweet @ Nov 13 2007, 06:18 PM)
yeah so...don't ever use a nanodrop to OD your samples............ closedeyes.gif ph34r.gif ph34r.gif ph34r.gif ph34r.gif

how many ul are you using with the nanodrop? you may not get reproducible, accurate results if you use less than 1.5ul.

another thing that could slow growth is contamination.

are you sure that your overnight grew as much as they usually do?


QUOTE (mdfenko @ Nov 14 2007, 07:19 PM)
another thing that could slow growth is contamination.

Yes, glassware must be thoroughly cleaned. Any detergent adsobed to surface of the glassware will cause the culture to grow slowly and even outright fail


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