I have a small flask with ~100ml of Amp resistant bacteria in LB [Luria-Bertani] broth, that has been at 4 degrees tightly sealed for 26 days. I need to grow a larger culture of this same bacteria and use it in inducible protein production.
Can I use my 26 day old culture as a starter in the larger volume?
That is not recommended and not standard practice. Bring the broth to room temperature, confirm viability in similar broth and agar. Re-prepare the culture in the broth in which the original culture was stored. I prefer 18-h broth culture, probably because I work mostly in production researches.
There is some risk that something else has grown, as the Amp has been degraded over time.
Try to regrow your bacteria from a small aliquot, as Maurice Ekpenyong has suggested. If it does not work (very likely), you could try a PCR on an aliquot in order to rescue the insert or a plasmid prep to rescue the plasmid.
If you still have the plasmid, I think it's safer to start over.
i agree with Maurice and Wolfgang. I would sub (plate out), confirm with colony PCR and then expand out a positive clone.
Well every body I did use a liquid culture that was 26 days old and I did get back good sequence data.
I did an overnight rejuvenation at 1:1000 inoculation and twice the normal resistance antibiotic. I used that for the starter to make a 1liter culture for inducible protein
I sequenced the BL21 bacteria following the protein induction and that was perfect and low background. And I got good protein out of the next phase.
And you are all correct about this being substandard practice, so far as standards go, but I'm keeping my protein.
Hey, if it worked, it worked!
Sometimes the only way to know if you can still use something is to just try it and see what happens. I know I've done quite a few "non-standard" steps in protocols (like digging a gel out of the waste container to salvage a band, sticking Arabidopsis seedlings in the refrigerator for a few weeks before transplanting, etc.) and it's often quite fine.
I'd recommend that you make a glycerol stock for long-term storage :0)
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