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Plasmid stability in Ecoli during storage


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#1 NielsDammes

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Posted 24 May 2013 - 08:57 AM

Hello everybody,

I had some questions about plasmid stability in E. coli cells. I though thatt I knew more or less how to store them temporary but apparently not.. I had a pUC19 plasmid with a 7 Kb construct which is very important for my activities. I prepped the plasmid and this yielded at least 500 ng/ul (eluted in 30 ul) which is a nice yield of course.

I kept the culture in the fridge and to be sure not to lose it, I made a back up by centrifuging a freshly grown culture and I stored the pellet at -20. Of course this is not a permanent solution but it seemed to me stable enough to keep it like that for the next 2 months. I was planning anyway to do some glycerol stocks in 6-7 weeks so I kept it for the moment like this.

This week I ran out of prep material so I re-innoculated some medium with my liquid culture from the fridge, which was standing there for max 5 weeks. The next day, I quickly wanted to measure the yield in order to estimate how much I should digest and somehow the nanodrop said that there was almost no DNA. Of course I didn't believe this and I put the whole tube on gel but without any visible bands. That evening I tried to innoculate it again and I also innoculated some media with a little piece of the pellet from the -20. The next day, unfortunately the same 'yield'... I used the second time a different miniprep kit but apparently there was no plasmid DNA there..

What I don't understand is, how can a plasmid be lost so quickly in both +4 and -20 storage? And above that, how is it possible that my cells grow in Ampicillin containing media when they don't carry the plasmid? During the second attempt I also innoculated some standard Ecoli cells and those didn't grow which means the antibiotics are working fine...

What on earth is happening here and how should I prevent this? Should I next time always make -80 stocks, even if I need some plasmids only for some experiments of a few weeks or is there an alternative?

Thanks!

Niels

#2 hobglobin

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Posted 24 May 2013 - 12:38 PM

as far as i remember when the antibiotics are gone then bacteria get rid of unnecessary plasmids quite fast, and after 5 weeks it's quite probably no antibiotic left
I'd suggest to do a stock culture at -20 degrees using glycerol http://ecoliwiki.net...l_Freezer_Stock
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#3 bob1

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Posted 24 May 2013 - 05:38 PM

Or jus make a glycerol stock at -80 the first time you grow up the bugs for plasmid prep.

#4 leelee

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Posted 24 May 2013 - 07:50 PM

Or jus make a glycerol stock at -80 the first time you grow up the bugs for plasmid prep.


This. It takes the same amount of time to make a glycerol stock as it does to spin down and freeze a pellet.

It is a good idea to keep a backup stock of everything you make anyway, you never know when you may need it again (even if unlikely).

Edited by leelee, 24 May 2013 - 07:50 PM.





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