Problem with using overgrown replica LB plates? - (Aug/06/2012 )
I left a replica LB Amp plate in the 37C over the weekend, and I'd like to know if they're still good to use. I've started liquid cultures but if they grow, can I use the minipreps without resequencing the whole plasmid? It's a replica plate, so I don't necessarily need single colonies, and satellite colonies are not an issue. I am primarily concerned about mutations that may have accumulated. Any ideas?
What are you planning on using the mini prepped plasmid for?
And I don't understand what you mean when you say that satellite colonies are not a concern because this is a replica plate.
They were replica plated while a colony pcr product was submitted for sequencing. I forgot about the replica plate over the weeken and didnt mark the oroginal colonies on the ligation plate. Now i want to now expand the clones with the correct sequence but I'd like to know if being on a plate for 3 days at 37c is cause for concern. Most things I've found about overgrown plates are warnings about satellite colonies but obviously for me if it grows then that's great. Rather I'm worried about potential spontaneous mutations in the backbone that could accumulate from 3 days growth or something else I'm potentially not thinking about being a problem
Ok then. I disagree that satellite colonies aren't a concern then, or that you don't need single colonies. In both cases you could end up with a mixed population of the clone you want and one you don't.
I would take a colony from your overgrown plate and re streak out onto a fresh LB Amp plate and grow it up from their. Better to do it right from the start then continue on your way only to find out later you don't have what you think you have!
As for the risk of mutations, my feeling is that the risk is no higher than during normal subculturing of your clone- but I could be wrong.