Re: [DIYbio] Freezing cells with plasmids

The competent cells I make in-house out of XL1-Blue cells in TSS buffer last a month with the highest competence within the first week if stored at -20 and I get a few colonies if using pure plasmid (miniprep) at the 5-week mark. The TSS recipe I use is a 2x solution of the following:

85% LB Broth (Miller Mod)
10% PEG (w/v using Miralax)
5% DMSO (v/v)
50mM MgCl2
pH 6.5

I grow 5mL of blank XL1-Blue cells overnight  in LB with 12.5mg/L tetracycline (genomic resistance) shaking at 37C, then inoculate 1mL of the culture in 120mL of LB-Tet (same conc) and shake until I reach an OD600 of 0.5 using my 3D printed turbidity meter. I then dispense the culture into as many 15mL tubes as my blood lab centrifuge (fixed angle 3500rpm standard clinical centrifuge CHEAP) can hold and place the tubes in a deep ice bucket for 15 minutes.

I then place the tubes in my centrifuge which I keep in my fridge at least four hours before and spin for 10 minutes.

I immediately place the tubes back on ice. Next I dump out the supernatant (liquid phase of tube) and pipette any remaining LB off the pellet. Next I take 1mL of ice cold LB and dispense it into one of the tubes containing my bacterial pellet, resuspend completely, and take the contents of that tube and transfer it to the next and resuspend. Continue until all the tubes have been resuspended and transferred. You will now have one 15mL tube containing around 1.2mL of heavily concentrated bacteria. Using a pipette, measure the exact volume of bacteria and dispense it to an empty tube.

Aseptically transfer equal volume of ice cold TSS into the suspended cells. Resuspend by pipetting up and down. Allow the tube to sit on ice for 10minutes to recover.

Distribute 100uL per 0.5mL PCR tube and freeze at -20. I use an old 0.5mL PCR machine (biometra uno) as my heat shocker (90sec at 42C) and (5min at 37C for agro). I prefreeze around 20 0.5mL tubes for an hour or so to not let the cells warm at all and then quickly place them in my freezer.

I get the absolute best efficiency, obviously, when using the cells that are just freshly made but they hold up alright for at least a month. My new chest freezer actually averages -30 so it buys me some extra weeks. More often then not I avoid complex ligations so all I really need is one colony. Anywho, that's my wacky protocol and it works fine for me.

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D

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From: Katherine Gordon
Sent: ‎6/‎27/‎2014 7:11 PM
To: diy...@googlegroups.com
Subject: Re: [DIYbio] Freezing cells with plasmids

I think that it is not the freezing but the thawing out that is hard on cells. No idea how long your cells will last because different cells have different life spans. I would like to know more of what you are doing, the type of cells used, collection methods and your reasoning or expected outcome.

thank you

On Fri, Jun 27, 2014 at 4:06 PM, Koeng <koen...@gmail.com> wrote:

Hey

I wanted to try out freezing cells in a -20 freezer at 10% glycerol a few days ago. So I did it, and it looks like they are frozen solid. I don't know much about freezing cells, but I assume since they are solid they should stay good for longer. I forgot to check the ones with 40% glycerol, but perhaps they aren't frozen

Has anyone tried this? Does anyone know if cells that are frozen solid will degrade over a long period of time? Of course I am trying it, I have cells in 10% 20% and 40% glycerol in both -20 and -80, but I'd like to know if anyone has experience with this. In a month or so I'll try to recover some plasmid from them. I'll just keep trying each couple of months and if it's working I'll start doing it ever 3 months. I am putting it directly into liquid culture, so all I am testing is if there is still plasmid, so I don't know how useful the results will be

Anyone have any experience with this? (BTW NEB says that their comp cells become less competent in -20, but did they freeze them solid? Is there a difference? Sorry, I am no expert at this, so that's why I am trying it)

-Koeng

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