picking up colony - (Jun/27/2005 )
I don't know whether you have experienced this, but it had happend to me several times and today happened again:
I picked up E.coli colonies and resuspend it in 5 ul of H2O, then I used 2 ul from it to do a PCR to verify my construct. Those colonies that contained the desired construct were cultured in LB broth with proper antibiotics overnight. Strangely sometimes it did not grow at all!!!
Any suggestions? this is really really nightmare
-bullfrog-
QUOTE (bullfrog @ Jun 27 2005, 06:22 AM)
I don't know whether you have experienced this, but it had happend to me several times and today happened again:
I picked up E.coli colonies and resuspend it in 5 ul of H2O, then I used 2 ul from it to do a PCR to verify my construct. Those colonies that contained the desired construct were cultured in LB broth with proper antibiotics overnight. Strangely sometimes it did not grow at all!!!
Any suggestions? this is really really nightmare
I picked up E.coli colonies and resuspend it in 5 ul of H2O, then I used 2 ul from it to do a PCR to verify my construct. Those colonies that contained the desired construct were cultured in LB broth with proper antibiotics overnight. Strangely sometimes it did not grow at all!!!
Any suggestions? this is really really nightmare
I don't know how u inoculated the colonies to LB broth,I often use toothpicks which are sterilized in advance.Generally it grows well,but sometimes it seems like have no bacteria in LB broth.My answer is that the colonies don't proliferate enough to make u see them through eyes,if u continue culturing them, u will catch them,best wishes!
-pfy1982-
If possible, switch to Kanamycin, as it is generally better and more selective than Amp. Contamination and satellite colonies are always a possible cause. Hope this helps. 
-k81878-