E. coli colonies didn't grow up in liquid culture. -HELP- - (Jan/29/2009 )

Hi there,

I have had trouble my cloning for more than 9 months…my project has not progressed because of this cloning trouble. However I could find colonies inserted the gene of interest (I checked them with colony PCR) after transformation, they did not grow up in liquid culture. I used several types of competent cells, vectors, media and changed culture conditions, but they didn’t work. Now I am trying to grow up colonies in LB media (the competent cell strain is DH5a). The insert size is about 600bp and vector is 5,000bp. It has not worked so far. I am almost crazy….
I am most grateful if you could give me some advises!!!

As a check, did you use primers that amplified across the junction between your 600bp insert and 5kb vector? Colony PCR is sensitive enough to amplify leftover DNA from the ligation that is sitting on your LB plate. So if you used primers that amplified only the insert, you can easily get a false positive signal from leftover DNA sitting on the plate.

If this is not a problem, it should be noted that DH10a is a bit slow growing. So it usually takes a little longer than usual for the culture to reach saturation. Try going it longer.

However, given your story, I am concern. Try growing your colony on an LB plate to make certain it is really antibiotic resistant. Is your gene by any chance being expressed? And if so is the gene product toxic to e coli?

Hi perneseblue,

Thank you so much for your comments! I used primers that amplified the insert and amplified whole MCS in the vector. I used these primers as a double checking.
I plated colonies that I got after transformation onto another LB plate, it grew up...
I also tried to incubate more than 2days in liquid culture, but it didn't work....

Cheers,
Rintaron

rintaron on Jan 29 2009, 01:38 PM said:

regarding the media you used: Are the plates and the liquid media from the same LB batch? Could it be that there is some contamination in the liquid media that hinders growth. You possibly could test it by making a "normal" overnight culture with E.coli (without any plasmids). If E.coli doesn't grow than something is wrong with the media.....

Cheers!

Hi Wolverena,

Thank you so much for your advice. But same media works for other clonings. I guess it's difficult to think about problem in this media. I used several types of media and cultured normal time, but it didn't work.

Thanks,
Rintaron

this might insult your intelligence, but check that the plasmid your using is conferring the right resistance to the antibiotics. in otherwords, make sure you're not putting tet resistant bacteria in ampicillin media. also, if your colonies are growing on LB only plates (no antibiotics), try to grow those cells in LB only media, that way you eliminate the possibility that there are some contaminants in your incubator thats preventing growth.

all in all, it seems that you are lacking a positive control, do a transformation with a plasmid that you know will grow and do the liquid media side by side with it. It will greatly help determine where the problem is.

another potential insult. have you made liquid cultures before?

in case you haven't, are you trying to grow a large volume from a single colony?

you should not inoculate more than 10ml with one colony. grow that out then inoculate a greater volume with that.

Hi pcrboy and mdfenko,

Thank you so much for your comments, Now I need any kinds of advice, thanks for your consideration.
I have experiences to grow a large volume from a single colony. I checked the right resistance to the antibiotics.... but I haven't checked about the tet resistant bacteria. Once I tried to grow up without antibiotics, but they didn't grow up as usual....

Have you tried just empty vector and see if it grows ? You might have told this in the forum but i just wanna make sure that u know the plasmid is really the EMPTY vector.

If growth with empty vector but no growth with the new construct

then it might mean that the gene is doing something fishy to you E.coli.

If you would want to do plasmid prep. You could actually skip the liquid culture. Just resuspend from the agar plate itself and proceed with the typical plasmid extraction method be it kit or conventional. that should do

Let us know what happen@

Cheers

rintaron on Jan 30 2009, 01:02 PM said:

Just because this has worked in the past for you doesn't mean it's ok. Have you tried making a starter culture first? If not, what do you have to lose except 8 hours or so?