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How to process your Addgene plasmid (bacterial stab)

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.

  1. Streak Bacteria for Single Colonies - use your bacterial stab to streak bacteria onto a plate, grow overnight, and isolate single colonies.
  2. 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.
    1. Addgene recommends selecting and inoculating 2-4 separate colonies for plasmid screening and verification.
  3. 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.
  4. After isolating plasmid DNA, Addgene recommends verifying the plasmid by diagnostic digest or sequencing before beginning any experiments.
    1. Perform a Diagnostic Digest - verify backbone and insert sizes
    2. 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”
    3. 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.
  5. 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.
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