A year at -20... does it work?

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Koeng

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Jun 29, 2015, 3:51:31 PM6/29/15
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Hey guys

A year ago I began an experiment to see how I could store things long term in a DIY manner. The results (note: poor controls. Please don't sue me and take all of this as opinion)

-Plasmids are best stored in E coli stabs
-E coli strains are best stored at -20 at 40% glycerol

Simply put, I put 3 things in the freezer for the year: bacteria culture with 10% glycerol, 20% glycerol, and 40% glycerol. I also put in a stab where I just picked a colony and stabbed it into a tube, normal method.

For the 10%, even though there was clear settling of cells I scraped from the top because the culture was frozen. No colonies appeared on the plate. I did the same with 20% and 1 colony appeared. 40% was not frozen so I just took a tiny from the bottom as well when I streaked and got surprising results

Although I used 1/5 of the small pellet at the bottom, I got *significantly* more colonies than I got with streaking out the entire stab. However, nearly 1/3 to 1/2 of these cells no longer held the RFP plasmid even though I plated on amp. On the stab plate, however (same batch of plates), I did not get a single white colony. There were less colonies, but still about 50 colonies.

So, overall, if you wanna store plasmids without an expensive freezer, store them as stab cultures. I can vouch that it works. (however sometimes mutations can occur in the genomes of the cells in stab cultures http://www.ncbi.nlm.nih.gov/pmc/articles/PMC516597/ )

-Koeng

Patrik D'haeseleer

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Jul 2, 2015, 3:54:56 AM7/2/15
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Excellent! Are you going to continue the experiment? If so, consider adding some tubes with 10% skim milk as well:


Patrik

John Griessen

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Jul 2, 2015, 3:32:30 PM7/2/15
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On 06/29/2015 02:51 PM, Koeng wrote:
> -Plasmids are best stored in E coli stabs
> -E coli strains are best stored at -20 at 40% glycerol
>
> Simply put, I put 3 things in the freezer for the year: bacteria culture with 10% glycerol, 20% glycerol, and 40% glycerol. I also
> put in a stab where I just picked a colony and stabbed it into a tube, normal method.

So, is that last one 0% glycerol? (stabbed it into a tube, normal method.)

>
> For the 10%, even though there was clear settling of cells I scraped from the top because the culture was frozen. No colonies
> appeared on the plate. I did the same with 20% and 1 colony appeared. 40% was not frozen so I just took a tiny from the bottom as
> well when I streaked and got surprising results
>
> Although I used 1/5 of the small pellet at the bottom, I got *significantly* more colonies than I got with streaking out the
> entire stab.

Does the sentence above mean: using 40% glycerol cells from the bottom, "*significantly* more than streaking
out the colony stabbed into a tube, normal method."?

> However, nearly 1/3 to 1/2 of these cells no longer held the RFP plasmid even though I plated on amp.

Infer that they mutated while growing just before you froze them...

> On the stab plate, however (same batch of plates), I did not
> get a single white colony. There were less colonies, but still about 50 colonies.

Was a white colony the kind you had hoped to preserve?


Nathan McCorkle

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Jul 2, 2015, 4:17:15 PM7/2/15
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On Thu, Jul 2, 2015 at 12:32 PM, John Griessen <jo...@industromatic.com> wrote:
>> On the stab plate, however (same batch of plates), I did not
>> get a single white colony. There were less colonies, but still about 50
>> colonies.
>
>
> Was a white colony the kind you had hoped to preserve?

I think not, since he was trying to keep the RFP plasmid around, which
presumably turns the colony red.

Koeng

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Jul 2, 2015, 4:24:35 PM7/2/15
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My apologies for being a bit vague.


>So, is that last one 0% glycerol?   (stabbed it into a tube, normal method.) 
Yep, it's in agarose in the refrigerator.

>Does the sentence above mean:  using 40% glycerol cells from the bottom, "*significantly* more than streaking 
out the colony stabbed into a tube, normal method."? 
Yes, I took a small chunk of the pellet from the bottom of the 40% glycerol tube. When plated, more colonies appeared than when I used the stab culture.

>Infer that they mutated while growing just before you froze them... 
I don't believe so. To be honest, I don't exactly know why they still grew. I think that it might be because of my plates, because these didn't look like satellites. \

>Was a white colony the kind you had hoped to preserve? 
I hoped to preserve, as Nathan said, the RFP plasmids. However, the white colonies still show the strain is good. 
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