Storing competent cells without -80c freezer

[William Beeson]

Hi all,

Any ideas on how to store moderate competency cells without a -80c freezer? I've read the cfu/ug goes down dramatically if stored at only -20c? How are you guys doing it?

Thanks,
-Will

[Sebastian S Cocioba]

I make em every three weeks. Store in -20C in TSS buffer. I make how much I normally use in two weeks plus minus a bit. It isn't a big deal. For super tricky ligations I make em fresh day of. Never missed a clone. How many colonies do you really need, though? 

Sebastian S. Cocioba

CEO & Founder

New York Botanics, LLC

Blog: ATinyGreenCell.com

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[William Beeson]

I just bought the "subcloning efficiency" competent cells from NEB (https://www.neb.com/products/c2988-neb-5-alpha-competent-e-coli-subcloning-efficiency) and they don't specifically talk about competency dropping for these cells, but for their ultra-high competency cells they have a FAQ (https://www.neb.com/faqs/1/01/01/can-i-store-competent-cells-at-20-deg-c-instead-of-80-deg-c) that says after 1 week of storage at -20C the efficiency is decreased by 250x.

I don't need high competency.  If I have >1x10^6 CFU/ug that should work, maybe even a lot lower would work.  Have you ever checked to see if the efficiency of your cells were actually dropping over time?  I wonder if the drops are only for ultra high competency cells (>1x10^9 cfu/ug).

[Patrik D'haeseleer]

On Saturday, March 5, 2016 at 8:44:54 AM UTC-8, William Beeson wrote:

for their ultra-high competency cells they have a FAQ (https://www.neb.com/faqs/1/01/01/can-i-store-competent-cells-at-20-deg-c-instead-of-80-deg-c) that says after 1 week of storage at -20C the efficiency is decreased by 250x.

Nice data point! They say "cells lost 94.5% of TE after only 24 hours of storage at -20°C. Cells lost 98.9% of TE after 2 days, and 99.6% of TE after one week of storage at -20°C" - so that would be 18x after one day, 91x after 2 days, and 250x after 7 days.

Anyone know whether electrocompetent cells maintain their competence any better, or whether there are any particular strains that tend to maintain competence longer when stored above -80?

Patrik

PS: Of course, all this also points out how important shipping conditions are likely to be for competent cells

[Sebastian S Cocioba]

Im just confused as to why people buy competent cells when you can easily make them yourself. Biohackers don't have money but have time so why not get a nice plasmid producing strain and make a TSS buffer based competent cell system. Mine last for 3 weeks in -20C. Works fine. I use a line of NEB turbos I bought like three years ago. Unless you have a -80 the price of comp cells are not worth it at all. Its not magic in those cells, its cell state and buffer used...all which can be obtained by the user. Commercial comp cells are just convenience for labs that absolutely need highest efficiency...though for hacker-spaces and personal labs I see no purpose in massively competent transformations. Its one of those fetishized metrics people rave about but its intrinsically pointless for most cases. After three weeks I still get 40 colonies which is more than enough to screen a ligation or colony pcr. Pardon the rant but I really don't see a point in buying when there are so many good protocols for making them simply. 

Sebastian S. Cocioba

CEO & Founder

New York Botanics, LLC

Blog: ATinyGreenCell.com

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[djwr...@gmail.com]

Which protocol are you using?

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[Sebastian S Cocioba]

I got made my own protocol as a spin-off of several. Streak cells onto LB overnight, grow single colony in 15mL LB overnight, take 1mL of overnight culture and Grow cells to OD=0.5 in 125mL of LB shaken for 4hrs-ish (NEB Turbos are fast!) at 37C 200rpm 20mm orbit in a 250mL erlenmeyer flask with foam plug, spin down at 3500rpm for 5 min in 8 15mL tubes full up. Decant completely, remove all the excess liquid with a pipette (very important to do so but do not disturb the pellet). Resuspend one of the 8 tubes with 1mL cold LB, take the entire resuspension into another spun down tube, use the liquid from the previous resuspension to resuspend the second tube. Keep doing this until all the pellets are resuspended and concentrated into one tube. Resulting suspension should be about 1.2mL. Add that volume of ice cold TSS 2x to the tube and mix gently by pipetting up and down. Dispense 100uL into sterile prechilled pcr tube. Makes about 24 reactions worth. TSS is transfer and storage buffer as per the original article on said buffer. I use lithium chloride instead of magnesium. Annecdotal evidence suggests it works better. Make sure to do all the above actions on ice and work quickly. Keep all tubes in contact with ice while resuspending. Using freshly miniprepped plasmid to test I get hundreds of colonies. I heat shock cells by incubating at 42C for 90 seconds in preheated pcr machine and then ice for 5min. Add 100uL SOC to recover if non-amp plates or tricky ligation, shake in pcr tube at 37C 200rpm in glass or plastic 60mm petri dish bottom so tubes rattle around for no less than 20min but no more than 40min. Plate 100uL per pre-warmed lb agar dish with appropriate antibiotics. Enjoy!

Sebastian S. Cocioba

CEO & Founder

New York Botanics, LLC

Blog: ATinyGreenCell.com

To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/E0B830AF-876D-494D-9080-7D7BB6C40A61%40gmail.com.

[djwr...@gmail.com]

Thank you😀

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[Patrik D'haeseleer]

On Saturday, March 5, 2016 at 12:06:11 PM UTC-8, Sebastian wrote:

Pardon the rant but I really don't see a point in buying when there are so many good protocols for making them simply. 

I do think there is an important tradeoff to be made there. I agree that in an ideal world, every biohacker should be able to make their own competent cells at pennies on the dollar. In the real world, we have many people who are still very much beginners when it come to lab techniques, and paying ~$10 per transformation for commercial competent cells can allow them to bypass a significant amount of extra labwork and get on with the project they really wanted to work on that much faster.

One could definitely argue that those people should learn to walk before they can run. By that same argument, one could argue that everybody should be put through traditional factory-school undergrad courses to learn the basics of wetlab and molbio techniques before they are allowed to do any transformations on their own. We prefer to throw people in at the deep end, and get them involved in projects right off the bat, even if that means taking a few shortcuts while they get up to speed with lab techniques. 

Sometimes it's also just a matter of practicality: if we just paid a couple hundred dollars to get a new construct synthesized at IDT, why not pay an extra $10 to transform that precious limited resource of DNA it into a reliable commercial competent cell line? Once we have a plasmid prep, we'll have plenty of DNA to go around, and the reliability and efficiency of the competent cells is far less of an issue.

So yes, we should definitely teach everyone to make their own competent cells. But that doesn't necessarily mean that commercially bought competent cells don't have any place in a community lab.

Patrik

PS: We also don't let people pour their own PAGE gels. That one's more of a safety issue, but you could make the same argument that everyone should know how to make their own cheap, custom gradient gels...

[William Beeson]

I want to echo Patrick's comments.  

Many people are limited in their time and their time has a definite and quantifiable value.  If it was possible to purchase and store competent cells for several weeks, rather than having to perform a protocol that requires multiple reagents and hands on time over a period of 24-48 hours -- that's a trade off many people will elect to make.  Many people routinely spend $10+ on coffee and breakfast, lunch, or a drink after work.  Even graduate students, which are probably near the lowest pay scale of anyone in the life sciences -- make approximately $15-20 per hour.  The "subcloning efficiency" cells I purchased were ~$70 for enough cells (six 400 uL tubes) to do about 50 transformations.  

I am storing the cells in my -20C freezer to see how they work 1, 2, 4 weeks out from the purchase date.  Will let you guys know what I find.

-Will

[Koeng]

Are these lucigen cells, in particular? I am interested to know how their storage goes. 

[Dennis Oleksyuk]

Will, it was 4+ weeks from your message. Any results of the storage experiment?

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[William Beeson]

Hi Dennis,

Thanks for the reminder!  I will let you know in a few days.  Already left the lab before I saw this email.  I actually forgot about this experiment so I will only be able to report the transformation efficiency at ~4 weeks.  I spent the money to buy these cells then did not do transformations.  I'll use the PUC19 vector purchased from NEB for the calculation.  I'll also make some aliquots of the tube I use and test again in a few days to see if a single freeze/thaw hurts the efficiency badly.

-Will

[William Beeson]

I performed the experiment.

I used the NEB subcloning efficiency cells.  They had been stored at -20C since March 5th (47 days, 6-7 weeks).  I didn't have much time, so I did the "fast transformation" protocol.  I used PUC19 vector also purchased from NEB.  I was plating onto LB + ampicillin.  I used 1 uL of the following DNA concentrations: 1000 ng, 100 ng, 10 ng, 1 ng, 0 ng.  Thawed the cells on ice, aliquoted 50 uL by pipetting into 2 mL eppendorf tubes chilled on ice containing 1 uL of the plasmid, incubated on ice for 3-5 minutes, heat shock at 42c for 30s, on ice for 2 minutes, add 450 uL of SOC, plate 100 uL onto the ampicillin.  

There was a very large number of colonies on the 1000 ng.  On the 100 ng there was ~110 colonies and on the 10 ng there was 6 colonies.  I didn't see any colonies on 1 ng or 0 ng.  Overall, I think the cells still worked ok.  The expected efficiency is ">1e6 CFU/ug PUC19".  I got ~6000 CFU per ug, but that was with the rapid protocol.

-Will