Freezing cells with plasmids
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Koeng
Jun 27, 2014, 7:06:42 PM6/27/14
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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
Katherine Gordon
Jun 27, 2014, 7:11:39 PM6/27/14
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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
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Koeng
Jun 27, 2014, 7:26:20 PM6/27/14
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I am freezing a strain of cells, DH5 alpha, that contain a plasmid, which I call "7". It is about 5200bp, and holds the gene for RFP. I inoculated 200µl of an overnight liquid culture into a centrifuge tube, then took 50µl of 80% glycerol and put that into the tube as well (variable amount of glycerol here. the example is for my 10% batch, I also a 20% and 40%). I then vortexed the cells, and put one batch into the -80 and one into the -20
I except the -80 cells to stay good for at least 6 months, but I am not sure about the -20s. I hope at least 3 months, because then I could begin storing strains with my plasmids at my home lab, which will significantly help me since I've found fresh DNA to usually be better than old DNA in some of my experiments. Plus I can directly put that onto plates or liquid culture to send to people.
-Koeng
-Koeng
John Griessen
Jun 27, 2014, 7:33:29 PM6/27/14
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On 06/27/2014 06:06 PM, Koeng wrote:
> Has anyone tried this?
No, but I thought it was the rapidity of freezing that avoided
cells being stabbed/deformed by ice crystals by way of smaller ice crystals
in rapid freezing... If so, freezing in your -80, or liquid N2, then storing in -20 might get results.
> Has anyone tried this?
No, but I thought it was the rapidity of freezing that avoided
cells being stabbed/deformed by ice crystals by way of smaller ice crystals
in rapid freezing... If so, freezing in your -80, or liquid N2, then storing in -20 might get results.
Koeng
Jun 27, 2014, 7:35:49 PM6/27/14
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Ok next week I will try snap freezing the cells and then placing them into the -20
One thing though: Yeast comp cells are supposed to be frozen very slowly in order to preserve them... Like if you want to get cells you put them in a styrofoam box then place it in the -80. Wouldn't this allow more time for ice crystal formation?
Sebastian Cocioba
Jun 27, 2014, 7:42:05 PM6/27/14
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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
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
From: Katherine Gordon
Sent: 6/27/2014 7:11 PM
To: diy...@googlegroups.com
Subject: Re: [DIYbio] Freezing cells with plasmids
Koeng
Jun 27, 2014, 7:47:48 PM6/27/14
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Next week I think I'll try that as well, except this time -80 as a positive control for the experiment.
In my experience, TSS isn't very efficient (at all) I get around 10^5 - 10^6. Perhaps with electrocompetent cells this would be higher?
Btw does anyone know of the cheapest, coldest freezer I could get? My first freezer is getting a little cramped with primers and plasmids.
SC
Jun 27, 2014, 9:53:37 PM6/27/14
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Suggestion: When you try to grow your frozen cells, start them off in a small overnight culture without antibiotic. Then move them to a culture with antibiotic selection. That will give them a head start to produce the antibiotic-resistance proteins.
Panasonic has a nice, small -80 freezer for about 4K.
Nathan McCorkle
Jun 27, 2014, 11:29:25 PM6/27/14
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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®ion=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
-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®ion=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
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Koeng
Jun 28, 2014, 12:45:15 AM6/28/14
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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
Koeng
Jun 28, 2014, 12:46:09 AM6/28/14
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Actually probably less than a second. Very very very quickly, almost immediate
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Mega [Andreas Stuermer]
Jun 28, 2014, 7:43:35 AM6/28/14
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I once smashed e coli + pVIB in the -20 freezer and successfully ressurected them on fresh LB Amp after 4 months.
I think I froze just in sterile water, LB and with glycerol added. (got a little bit contamination with glycerol though on the amp plates, but 80% were e glowli)
maybe ny dirty procedure killed 99% of the cells but there were like 10000000 cells initially so I didn't care
Jeswin
Jun 28, 2014, 8:23:11 AM6/28/14
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cryoprotectant [1]. It prevents the formation of ice crystals. My
procedure is ~350uL glycerol (eyeball it in a 2mL microcentrifuge
tube) then adding 500mL fresh bacteria culture. I quickly vortex until
fully mixed and stick in the -80C. I just cultured some recently that
I stored away last year. Just quickly stab it and throw the pipette
tip in LB broth+Antibiotic.
[1] http://www.level.com.tw/html/ezcatfiles/vipweb20/img/img/20297/Cell_freezing_protocol.pdf
Jeswin
Jun 28, 2014, 8:23:57 AM6/28/14
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On Sat, Jun 28, 2014 at 8:22 AM, Jeswin <phill...@gmail.com> wrote:
> procedure is ~350uL glycerol (eyeball it in a 2mL microcentrifuge
> tube) then adding 500mL fresh bacteria culture. I quickly vortex until
oops. I meant 500uL culture.
> procedure is ~350uL glycerol (eyeball it in a 2mL microcentrifuge
> tube) then adding 500mL fresh bacteria culture. I quickly vortex until
DIY BIO Groningen
Jun 28, 2014, 4:45:29 PM6/28/14
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Which cells? E.coli can stand -20 with 10% Glycerol but better at -80 for periods longer than 6 months. In general you have to pellet down the cells by centrifugation at low speed (5000g) then discard the super and resuspend the pellet with LB and Glycerol or PBS and glycerol.
Alessio
DIYbio Groningen
Sent from my iPad
Sent from my iPad
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Richard Kramer
Jul 1, 2014, 4:41:17 PM7/1/14
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In my experience that much glycerol at -20 will not freeze solid instead it is just a very viscus solution. I had a plasmid preserved in DH5's for 3 years that I had mistakenly placed in the -20 rather then -80 and they grew just fine when used to inoculate a culture. The only problem was that most of the cells had settled to the bottom of the tube.
It is my understanding that at -80 with a 40% solution storage is nearly indefinite with a cell line let DH5's
.
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