Plasmid Handling and Storage

Addgene recommends the following steps to ensure proper handling and storage of your plasmids for future use. If there is a problem, please contact Addgene within 30 days of receiving your plasmid.

View Information For:

• Short-Term Storage
• Long-Term Storage
• DNA - Filter Paper or Tube
• Plasmid Purification
• Plasmid Verification
• Citations in Future Publications
• For information on kit storage, please check out our kit handling instructions.

Storage

Short-Term Storage – Store at 4 °C

Bacterial Stab

Bacterial Stabs

Addgene ships plasmids as transformed bacteria in stab culture format. A stab 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 up and out of the puncture point to spread across the surface of the stab culture. A picture of a typical stab culture with visible growth can be seen to the right.

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. You should plan to create a glycerol stock for long-term storage within 2 weeks of receipt.

Watch our video below for step-by-step instructions on how to store your plasmids.

DNA - Filter Paper or Tube

DNA on filter paper or DNA suspension in tubes should be stored at 4 °C upon receipt. You should plan to amplify the DNA for long-term storage within 2 weeks of receiving plasmids. Read below for more information on handling your DNA samples.

Long-Term Storage

Bacterial Stabs

Within 2 weeks of receiving your new Addgene plasmid you should create a glycerol stock. To create your glycerol stocks, please follow Addgene’s protocol below.

1. Streak Bacteria for Single Colonies - use your bacterial stab to streak bacteria onto a plate and isolate single colonies.
2. Inoculate an Overnight Liquid Culture - using a liquid culture will allow you to grow enough bacteria for plasmid DNA purification and for creating glycerol stocks.
3. Create a Glycerol Stock and Store at -80°C - most plasmids are stable for years when stored as bacterial glycerol stocks.

DNA - Filter Paper or Tube

Within 2 weeks of receiving your new Addgene plasmid you should create a glycerol stock. Read below for our general protocol for creating glycerol stocks:

1. Transform DNA into bacteria. Read our bacterial transformation page for more information.
2. Plate on an appropriate antibiotic agar plate and grow overnight. Ensure that you streak bacteria for single colonies.
3. Isolate single colonies and inoculate an overnight liquid culture.
4. Create a Glycerol Stock and Store at -80 °C - most plasmids are stable for years when stored as bacterial glycerol stocks.
5. Additional information can be found below in our section on handling DNA.

DNA - Filter Paper or Tube

DNA on Filter Paper

Your plasmids have been provided in the form of DNA on filter paper. Addgene provides you with two spots of DNA, a total of 10–50 ng of DNA, for each plasmid. It can be stored at 4 °C.

To recover your plasmid:

1. To recover the plasmid, use a clean razor blade to cut out one of the circles containing your DNA.
2. Immerse the circle in 30 µL of TE and pipette to mix.
3. After waiting for at least 10 minutes, use 2 µL to transform competent bacteria.

DNA Suspension in Tubes

Your plasmids have been provided in the form of DNA suspension in tubes. Addgene provides you with 10-50 ng of DNA for each plasmid. It can be stored at 4 °C.

Your plasmid has to be amplified before use. To amplify:

1. Use 2 µL to transform into bacteria. Read our bacterial transformation page for more details on performing transformations.
2. Follow manufacturer instructions for transformations into your choice of competent cells.
3. Plate on an appropriate antibiotic agar plate and grow overnight.

Plasmid Purification

After proper short and long-term storage of your new Addgene plasmid you may wish to purify the plasmid DNA out of the bacteria. Addgene has provided an example of a manual miniprep protocol for isolating plasmid DNA from an overnight liquid culture.

1. Inoculate an Overnight Liquid Culture - using a liquid culture will allow you to grow enough bacteria for plasmid DNA purification.
2. 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.

Plasmid Verification

After isolating your new plasmid, Addgene recommends verifying the plasmid before beginning any experiments utilizing it. Addgene recommends verifying your plasmid by diagnostic digest or sequencing.

Addgene has provided information on how to select the proper restriction enzymes for your digest as well as a general protocol and troubleshooting tips. We also have information for Selecting or Designing Sequencing Primers and Analyzing Your Sequencing Results. Please contact Addgene within 30 days of receiving your plasmid if there is a problem.

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 those Addgene has performed on the plasmid.

Search addgene.org for the catalog number of your plasmid for any recomended protocols from the depositor.

Citations in Future Publications

The plasmids you have received were created by your colleagues. Please acknowledge the Principal Investigator, cite the article listed on the plasmid datasheet, and include the Addgene plasmid number in future publications.

Contact Addgene

Thank you for requesting plasmids from Addgene. If you have questions or would like to speak with an Addgene scientist, contact us at .