4 months at -20

[Koeng]

Hey!

You might remember that a few months ago I posted on hypothetically storing E coli long-term at negative 20. After 4 months, I have verified that every condition I tried, 10%, 20% and 40% glycerol, all gave valid new colonies with the plasmid I had originally in them. It hasn't been a year yet, but this shows some promise for a technique DIYbiologists can use instead of storing DNA in plasmid form (I personally don't like doing that because its degraded for me before, or I don't have enough and the old never gets used, or I need a lot more and don't want to retransform)

Experimental conditions (rough layout. Plasmid 7 is an RFP plasmid I use)

1 culture of plasmid 7, with 10% glycerol

1 culture of plasmid 7, with 20% glycerol

1 culture of plasmid 7, with 40% glycerol

These 3 were placed in a frequently used -20 freezer box, and left for 4 months.

After 4 months, a small amount of liquid (~30µl) of each was put into new liquid culture, along with 1 from the -80 as a control (I should have done plates, sorry)

After 16 hours of growth at 37c with shaking these were analyzed for RFP expression.

RFP expression was confirmed. The cells, or at least a significant amount of the cells, had the plasmid and were growing.

Richard Kramer has similar experience with this from the last thread - https://groups.google.com/forum/#!searchin/diybio/20$20freezer/diybio/lJjLfMX4OK4/G3VpcKEFTtwJ

I also personally found that the cells tend to settle and that the liquid also stays viscus. I'll be continuing this experiment, but just wanted to give anyone that wanted to know a heads up that this is a viable way to store cells for at least 4 months, and reportedly 3 years. If anyone wants to try storing cells this way, or is storing them this way, give me a heads up so I can gather more data and replicates to give a definitive answer.  Thanks!

-Koeng

[Cathal Garvey]

Thanks Koeng! Reading the first paragraph I was thinking "Survival's
fine, but what about plasmid?"..only to read further and see that you
have that covered, too! :)

Nice work, thank you for following up with the rest of us.

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[Koeng]

It might be helpful to note that the cultures at -20 had less RFP/growth than the one from the -80, even though I added more cells to the -20s. So there is probably some loss of cells, but how much I don't know yet.

-Koeng