Lost in plasmid preperation - (Aug/28/2007 )

Hi all,

I have a problem of preparation of plasmid from E.coli DH5alpha, which transformed with plasmid carry Ampicillin resistance. I normally get well identified colonies of E.coli plus a few small colonies around them as back ground for the result of transformation. Then I pick the colony from the very center of the big colony for inoculation as starter culture 4 ml and make the Mini prep (rapid alkaline) out of it, so I recover the plasmid and get beautiful result. After keeping at 4 °C for 5-6h., the starter culture is then diluted 1/1000 for inoculation in 100 ml LB medium, The cells grow very well in selective medium, and the Maxi prep (rapid alkaline) is performed, but this time I can't recover the plasmid !!!

- Selective LB medium contains Ampicillin 0.1 mg/ml
- Same E.coli strain no problem with amplifying the plasmid carry Kanamycin resistance.
- When use Ampicillin as selection in this E.coli host, plasmid can be recovered only when the inocution sample is prepared from picking colony.
- Nothing wrong in plasmid isolation processes.


This really drives me nut Anybody could help me solve this mystery?

Thanks

This could be a problem with the ampicillin selection. Cells carrying the plasmid express the beta-lactamase enzyme, which inactivates ampicillin. This is the selective pressure that forces the cells to keep the plasmid. The problem with this system is that beta-lactamase is secreted into the medium by the cell.

This means that when you grow the 100ml culture, you start with beta-lactamase in the medium. This could break down the ampicillin in the medium, removing the selective pressure and allowing the cells to grow without carrying the plasmid. If this is the cause, then there are three ways to address the problem:

1. Before innoculating the 100ml culture, wash the starter culture in some antibotic-free LB to remove the beta-lactamase
2. Use a higher concentration of ampicillin. The satellite colonies in the plates also suggest that the ampicillin concentration you are using is too low.
3. Use carbenicillin instead of ampicillin - this is a tougher substrate for beta-lactamase so is not broken down so easily.

Thanks bitesizebio guy for the answer and suggestions. Those really help a lot for my experiment in future. I'm going to try out what you have suggested.

alternatively, you could perform several mini preps...
I've had a similar problem when doing maxi-preps, so I hate them. I rather do 10 minipreps (50ml of culture media at a time) at the same time.
I think it's a problem in the scalation process.

hi!

i once had the same problem with you... couldn`t recover plasmids from Ecoli after transformation. Our solution was to use a toothpick instead of loop to pick Ecoli colony for inoculation. The notion is that we wanted to make sure our Ecoli were not killed by heated loop [heat for sterilization], by using toothpick, we could change after every pick.

and...the result was good....we got plenty of plasmids!