Addgene ships plasmids as transformed bacteria in stab culture format. A stab culture is a type of Luria Broth (LB) Agar media, similar to a standard LB Agar plate. Unlike an LB Agar plate, a stab culture is created by piercing the LB agar with the bacteria instead of spreading it on the surface. The bacteria in a stab culture grow from the puncture site to spread across the surface of the stab culture.
Short Term Storage of Bacterial Stab
Stab cultures should be stored at 4°C upon receipt. The bacteria in the stab is guaranteed to live for at least 2 weeks when stored at 4°C.
Long Term Storage - Isolating and Verifying the Plasmid and Creating a Glycerol Stock
Within 2 weeks of receiving your new Addgene plasmid you should verify that the plasmid is correct and create a glycerol stock. Please follow Addgene’s recommended protocols below.
- Streak Bacteria for Single Colonies - use your bacterial stab to streak bacteria onto a plate, grow overnight, and isolate single colonies.
- Select an isolated, single colony to inoculate an overnight culture. Using a liquid culture will allow you to grow enough bacteria for plasmid DNA purification and for creating glycerol stocks.
- Addgene recommends selecting and inoculating 2-4 separate colonies for plasmid screening and verification.
- Isolate your Plasmid DNA - many companies sell miniprep or maxiprep kits for easy plasmid isolation. Addgene also provides a protocol for plasmid purification without a kit.
- After isolating plasmid DNA, Addgene recommends verifying the plasmid by diagnostic digest or sequencing before beginning any experiments.
- Perform a Diagnostic Digest - verify backbone and insert sizes
- Sequence your Plasmid - verify key regions of the plasmid using DNA sequencing and compare these to sequences Addgene obtained from the plasmid, found on the plasmid page under “Sequences”
- After screening 2-4 colonies, if you find that your plasmid DNA is not correct, please view the instructions on how to report a problem with a plasmid.
- Once you have verified that the plasmid DNA is correct, keep the glycerol stocks corresponding to the colony that was verified- most plasmids are stable for years when stored as bacterial glycerol stocks.