E.coli are growing in LB/Amp plates but not in liquid medium - (Jun/04/2007 )
Hi
I am doing a subcloning to change the expression vector of my insert. I am using DH5a cells. After the ligation and transformation, colonies were grown on LB/Amp plates and the inserts are confirmed using colony PCR with controls. However, the colonies I picked could not grow on liquid LB/amp. The medium was clear overnight! I tried several times but the results are same. There was a time that 2 out of 5 colonies did grow, but I could not get any DNA by miniprep. Im going crazy, Is there anything I was possibly doing wrong?
I checked my DH5a cells, they don't grow on LB/amp without plasmid. The Ampicillin concentration of both LB/Amp plates and liquid medium are 50ug/mL.
I appreicated your help.
Harvey
hi
it also happened sometimes to me too.
the colonies you got on lb plates may not be the ones you want.....I mean, they do not have the insert integrated properly.
i am not so sure what happen exactly, but when I run the colony PCR I got good bands as you did....I was told that sometimes, when you do transformation, some of the dna sample remain on the surface of the cells....DNa is quite resistant.....and it is that what you amplify brying y PCR.
Anyway....if you could not grow the colonies in liquid culture with antibiotics after several times, it means that the colonies you have are false positives and you need to do transformation again.
Good luck. 
How to explain the cells grow on LB/amp plate? It also grow when I streak them on other selection plate. They just don't grow in liquid medium.
It's really frustrating.
It's really frustrating.
it also happened to me. but after streaking i was successful to grow the e coli in liquid medium. check you medium or make fresh medium. do you use antibiotic from same tube that you used for making plate???
how old is your e coli plate when you tried to get growth in liquid medium???
It's really frustrating.
it also happened to me. but after streaking i was successful to grow the e coli in liquid medium. check you medium or make fresh medium. do you use antibiotic from same tube that you used for making plate???
how old is your e coli plate when you tried to get growth in liquid medium???
The antibiotics are from the same tube. The plates are 1 month old, do you think it may be the cause ?
I'll make new plates and give it a shoot.
In addition, how can I avoid false positive in colony PCR reaction?
I never purify my ligation mixture, and perhaps I was putting in too much inserts so I got false positive results.
your plates are pretty old, you should make fresh plate i think
I second that. The antibiotics is probably much degraded. lactams degrade quite readily when wet.
as for false positive results, always PCR across the junction between the insert and vector. Since this junction is unique, it is very unlikely that a random band (which sometimes appear in the PCR) will be the right size to be mistaken as a product band.