Re: [DIYbio] A year at -20... does it work?

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?