Re: [DIYbio] Freezing cells with plasmids

Makes sense. I don't actually fully thaw the cells, I just scrap a little off the top and inoculate into some liquid broth. Thawing takes seconds, so I think I might be able to get pretty high recover rate

-Koeng

On Friday, June 27, 2014 8:29:25 PM UTC-7, Nathan McCorkle wrote:

TL;DR; freeze slowly (-1C/min from starting temp to -30C, then
-0.5C/min to -50C, then -1C/min to -100C, then directly in LN2), thaw
fast(submerse in 37C water bath for 1-2 mins)

When I put cells in a -80 in school, we used a 'freezer buddy' (though
google only shows relevant results when you add the keyword
nalgene)... it was a plastic tube holder that was submerged in
isopropanol. The advertising schpeal says it provides controlled
1C/min cooling.
http://www.sigmaaldrich.com/catalog/product/sigma/c1562?lang=en&region=US


Remember cells can get cut by ice, but the forming crystal starts at a
single point, then grows enough and potentially bumps into another
crystal that also started at a single point. These crystals are not
just of water, remember the other stuff in the cell? Remember how ppl
also crystallize protein and DNA to study with x-rays? So the water
and other stuff starts crystallizing around water and other stuff...
it happens most of the 'stuff' in general is water. So water starts
crystallizing in an impure but mostly water environment. Now we get
into a calculus problem. As the ice crystal forms, the percentage of
water in the liquid phase decreases, and le chatlier's principle needs
called into mind. Le Chatlier's generally says that the higher a
concentration, the more likely that stuff is to do something. So now
that the ice has grow, it took water from the bulk liquid, and the
water decreased % wise, it decreased in concentration. Now the water
is less likely to add to the crystal (than before the first addition
to the crystal). Conversely, it is more likely for non-water to enter
the crystal.

I believe the idea with the freezer buddy is the opposite of why you
want to thermocycle DNA and primers quickly. In DNA thermocycling, a
faster transition yields more specific binding of primer to target
sequence. With a fast transition, there is little time for random
chance to strike... 'survival of the physics' happens... the molecules
that bind with the least energy remaining bind faster. In an ice
crystal, this would mean water would be the prime choice for a binding
event in short time (fast transition). This would push out all the
other 'stuff' in the cell, and form a huge chunk of pure water-ice
(water is again, not normally 'pure'/100% concentration in a cell).
This might also rip the cell open if the action ice expanded a lot, or
if during freezing, the ice completely absorbs any water and causes
osmosis to rip a membrane. If the cell thaws after freezing in such a
state, and thaws slowly, water concentrations will be much lower on
the partially-frozen core-side of the cell membrane, so all the
concentration gradients the cell had setup would be messed with. Too
much messing and the cell can't recover. So thaw quickly to minimize
the time for osmosis and similar 'crap' to happen :P


http://www.lifetechnologies.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/thawing-cells.html

(mammalian is usually more sensitive than others, but some species may
be special) https://unclineberger.org/tissueculture/general-protocol-for-the-cryopreservation-of-mammalian-cells

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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--
-Nathan