I have my plasmid (pCMV-4 backbone) in a DH5alpha cells and I have spread it on a LB+AMP plates (final conc of AMP= 0.1mg/ml). These plates have colonies that grew well with the expected shape and size. However, when I picked the colonies to do starting culture for Maxiprep, they didn't even grow at all. (clear LB Broth after 16hrs of overnight shaking at 37 deg celcius)

I have done this set of maxiprep before and didn't encounter any problem but I have no idea what has gone wrong this time round.

At first, my suspicion is the AMP cause my AMP was one month old. However, I don't think this is case as even if no or less amount of AMP, I should be able to see cloudy starting culture. My another suspicion is the autoclave conditions of the LB Broth. Stated on the bottle, the LB Broth should be autoclaved at 121 deg celcius for 15mins. However, I have done it for 20mins. May I know if this slight extension in duration of autoclave, does it have any significance in the nutrients of the LB Broth? I will try the 15mins first.

Is there any other possible reasons that my starting culture does not grow?