chloramphenicol. - what is going on now?! (Sep/14/2006 )
i received the plasmid to express the protein and the protocol says add ampicillin and Cm (i suppose it is chloramphenicol), so I add 20ug/ml as it says nothing about the concentration and couldnt find direct concentration anywhere. so i guess this should not completly inhibit the host growth but enough to make the plasmid ones grow better. i culture....no growth at all 
can't think of how to explain this. i am now culturing without Cm just to confirm that it was the reason cells didnt grow, but somehow i feel it was. did anyone experience something similar??? thanx a lot!
Usually you only need one antibiotic to select for the plasmid in bacterial culture, I would use Amp at 50-200 micrograms/ml. Cam is sometimes used for selection in mammalian systems.
Nope. Not really.
I have worked with plasmids with gives both chloramphenicol and ampicilin resistence. The concentration of chloramphenicol used is 20ug/ml going up to 30ug/ml (for the 30hr cultures). I use 25ug/ml of ampicilin.
So if your cells were truly ChloR, AmpR then they should grow, and grow very well too.
Is it possible to verify the presence of the chloramphenicol marker. Either by the plasmid's sequence map, marker PCR, digest, or the person who gave you the plasmid.
Oh by the way, is your beta lactamase gene the standard one? I understand that for some variations of beta lactamases there is a synergistic interaction between chloramphenicol and ampicilin. I can't quite remember what happens if lots of ampicilin is used.
the srtange thing is that i received that plasmid and poeple who worked with it used Cm with no problem. 
so that means the plasmid should be resistant to Cm no? 
how come it doesnt grow....could it mean I dont have a plasmid??? i electroporated it and got a lot of beautiful colonies which were dilution depedant. what do you think?
hi
i think they mean to grow the culture overnight with ampand then artificially enrich in plasmid by adding chloramphenicol.
If so, here is my protocol :
- grow an overnight preculture
- inoculate 100ml by 100µl of the preculture
- grow to OD 0.7-08
- add 73µl of a 34mg/ml chloramphenicol solution
- grow overnight at 37°
i think they mean to grow the culture overnight with ampand then artificially enrich in plasmid by adding chloramphenicol.
If so, here is my protocol :
- grow an overnight preculture
- inoculate 100ml by 100µl of the preculture
- grow to OD 0.7-08
- add 73µl of a 34mg/ml chloramphenicol solution
- grow overnight at 37°
Fred_33, might you know how much more plasmid does one get with chloramphenicol treatment compared to the absence of such treatment.? A rough number would do. Thank you.
Fred, i got their protocol step by step and it says:
-incubate colony for 12 at 37 (amp+Cm) in 3 ml LB they dont mention the concentration of Cm however.
-Put those 3 ml into 300 ml (Amp+Cm) and shake overnight at 37.
-Put 30 ml of the preculture into 660ml of LB (Amp but no Cm) and incubate until OD reaches 1. for protein expression. IPTG etc....
however i think your suggestion is more reasonable. First one would grow the culture and then inreach it...how can I inreach it if it is not growing??? 
but i just want to make sure, it has nothing to do with the presence or absence of plasmid right?
ok now my PI says it is because i didnt grow cells on Cm+Amp plate. just the one containing Amp. so I didnt pick up the colony which contains the needed plasmid, that is why it didnt grow on Cm plate. 
Strange...
could you describe your plasmid? Is your cell carrying two plasmid? Is the E.coli strain naturally AmpR?
If the plasmid carries both resitence genes for Amp and Cm, then it wouldn't matter which marker was selected as it would have pulled the whole plasmid along for the ride.
Anyhow, on the off chance something is happening, restreak the cells on Amp+Cm and see what grows. Or if it is faster, retransform the plasmid and plate on Amp+Cm plates. However do investigate that the identity of the plasmid you were given. It is probably right but you need to know for certain.
it is pGEX 4T-1 i think low copy plasmid, which has a resistance to Cm. I think all pBR322 derived plasmids have some resistance to Cm, and they are low copy...anyway i will streak it again on Cm+Amp plate, but i can't see how could I have picked a "wrong plasmid" if it grew on Amp plate.