Creating Bacterial Glycerol Stocks

Racheal, a Lab Tech here at Addgene, shows you how to create a glycerol stock solution to store your plasmids indefinitely.
For the full protocol text, visit www.addgene.org/protocols/crea...
Bacterial glycerol stocks are important for long-term storage of plasmids. The addition of glycerol stabilizes the frozen bacteria, preventing damage to the cell membranes and keeping the cells alive. A glycerol stock of bacteria can be stored stably at -80°C for many years.
This protocol begins after you’ve already created a bacterial culture, so please see out video on Inoculating Liquid Bacterial Culture.
www.addgene.org/protocols/inoc...
___________________________________________________________________________
0:00 Intro & Overview
1:22 Materials
2:27 Creating the Bacterial Glycerol Stocks
3:39 Recovering Bacteria from your Glycerol Stocks
___________________________________________________________________________
Featuring: Racheal Komuhendo
Written, Directed, & Edited by: Quintin Marcelino
Music by: Lauren Duski, Vibe Mountain, and RKVC

[@addgene]

Addgene does not regularly monitor comments posted here, so we may not see your question immediately. We’d be happy to answer any questions sent to [e-mail] as soon as possible. Please include the name of the video along with any questions so our support team can help. Thanks!

[@rkomukyeya]

That’s my sister👏🏿👏🏿💓

[@AsadAli-to2zu]

Thank you Racheal.
I already knew the process. But I like your video and your style of explaining things. Keep up the good work.

[@coralyscintroncintron8131]

Thank you for your videos! They are easy to follow and well explained.

[@saakshisaini4920]

Thanks for sharing this protocol.. also you are a deloght to watch.. thank you very much!😊

[@davidcristianrodriguesluca3496]

Thanks for this video. It has been helping me a lot.

[@udayawimalasiri5957]

Informative and nice presentation. Thank you!

[@park_chaeni]

Thanks a lot from KOREA!

[@subashdevkota207]

Thank u for beautiful tips

[@DrHKM-ui9xn]

Thanx for this important info

[@Val-zx7ro]

Wonderful!

[@mhamado.abdullah6925]

Well done,

[@mwebesaaaron1811]

Thank you.

[@shameeramahawattage7150]

Thank you

[@hanielnkadi3040]

Thanks a lot

[@rahmankarim8846]

thank you

[@alejandrogilgomez3853]

Thank you for the video. I think the 70% isopropanol in the materials needed list may be a mistake since it is no longer mentioned in the rest of the video.

[@derinkamal7284]

Thanks for your Amazing Video, I have Clostridium Species, I want to Save it, Which Protocol do you recommend,?? Can I use same protocol that you talked abouted on the Video??

[@imatg01]

Wish my teachers were like u rachell
☹️

[@chaeranichaerani5416]

Thanks for the clear explanation. I have 2 questions. Does the glycerol have to be sterilized first? What is the 70% isopropanol for?

[@addgene]

Thanks for the question! If your stock solution of glycerol isn't already sterilized, you can sterilized a diluted glycerol solution (ie. 50% glycerol) using an autoclave or HEPA filter.
The 70% isopropanol is used to help keep your work area sterile.

[@tehminaahsankhan182]

hi can we store our glycerol stocks at -20. will bacterial stocks stored at -20 be functional.

[@asimabibi1141]

Superb,please take up the topic of micro manipulation

[@nurulnadhirahruzelan798]

hello, so nice video. I would like to ask. is it okay if I streak my bacteria onto the nutrient agar instead of LB agar?

[@addgene]

Thanks for the question! Nutrient agar can also be used. In general, LB agar is a subtype of nutrient agar. Nutrient agar is very rich and can grow a large variety of bacteria.

[@kksanshine1706]

Thank you so much❤️ l have some question. Can I stored E. coli stock in -20 c instead?

[@addgene]

Thanks for the question. Unfortunately, no. The glycerol/E. coli mixture would not freeze completely at -20, which is why lower temperatures are required for long-term storage.

[@fadiaaljanabi4054]

thank you
can I use the same concentration and protocol to stock staphylococcus species and use it then .
and for overnight bacterial culture i must preper it by use LB broth?
thanks a alot

[@addgene]

Thanks for the question! Different strains often require different growth and storage conditions. If you are working with S. aureus, I would recommend following this protocol from Current Protocols in Microbiology:
www.ncbi.nlm.nih.gov/pmc/articles/PMC6211185/

[@nirvanasingh2]

Should the 50% glycerol be sterilised/autoclaved?

[@andreasstuermer4946]

Does the overnight culture that's diluted with glycerol contain the selection antibiotic? Or should you leave that away?

[@addgene]

No, you don't need to add antibiotics to the glycerol stocks.

[@swati656]

please share the procedure for fungi as well

[@addgene]

Thanks for your question! Check out ATCC’s mycology guide. It has sections covering cryopreservation and lyophilization of yeasts and filamentous fungi. www.atcc.org/en/Guides.aspx

[@kaneezehussain5039]

Please tell that the bacterial culture should be an overnight culture? Or we can also use a 3 day old liquid culture?

[@addgene]

Hello, we recommend incubating liquid cultures overnight. For the best results, we recommend using your liquid culture immediately after. Please see our protocol for Inoculating a Liquid Bacterial Culture. www.addgene.org/protocols/inoculate-bacterial-culture/

[@hamzaalati4959]

Hi, Dear, I have a gene in plasmid bacteria in -80 and I have taken some of it and I did miniprep and so on so before I have done miniprep I took 2 ml from that before I did centrifuge and miniprep and now I need to take the 2 ml and I need to return that to -80 Could I do this by puting it in bacteria and glycerol and return it in -80

[@addgene]

You should be able to use the additional 2ml to make a new glycerol stock. You can check out our written protocol for a little more info: www.addgene.org/protocols/create-glycerol-stock/

[@hussainzahra2447]

Thank you I have acquisition can I use brain heart broth instead of lb for preservation of E.colo

[@addgene]

Thank you for your question! While our laboratory uses LB for growth of E. coli, it may be possible to instead use BHI. Please see the following publication (pubmed.ncbi.nlm.nih.gov/26120385/), which should provide some additional information.

[@hussainzahra2447]

How much amount of media to glycerol should I mix and when I should add glycerol to broth before or after bacteria have been grown

[@addgene]

@@hussainzahra2447 Hello again! You should add glycerol to the culture after it's been allowed to grow overnight. We recommend combining 500 uL of your overnight culture with 500 uL of a 50% glycerol solution. You can find more details and tips for creating a glycerol stock in this protocol - www.addgene.org/protocols/create-glycerol-stock/

[@hussainzahra2447]

Addgene thank you so much

[@TalsBadKidney]

2 cool

[@designerguy6257]

Hello. I am writing a science fiction graphic novel where a scientist finds a plant whose cell can express a phenotypical gene after not being able to find other plants that can express the gene. The gene is to take a human gene, transect that gene to a plant and then transect that transformed cell into another human to be expressed with a particular DNA. I wrote a scene where the scientist has discovered the correct plant that can express the gene that needs to be expressed without being too toxic to the plant. The only catch is he does this in nature by himself. Somewhere in the wilderness. He escapes from a lab, grabbing minimal essential tools to perform the experiment. But he later finds, on his path in the wilderness, a bunch of flowers that can express the gene he needs to transect without it being too acidic. Is a scenario where a scientist can do something like this outside, or in a sheltered area by himself, maybe a gas station or something?

[@desitraveller4282]

Thank you