You have just received your kit from Addgene. Now what?
Addgene recommends the following steps to ensure proper handling and storage of your kit for future use. If there is a problem, please contact Addgene within 30 days of receiving your kit.
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Kits Shipped on Dry Ice
Store the glycerol stocks in the kit at -80°C upon receipt. Take precautions to minimize freeze-thaw cycles. Keep the plate on dry ice to prevent thawing. To access a plasmid, use a sterile pipette tip to puncture the seal above an individual well and then streak a portion of the glycerol stock onto an agar plate to isolate single colonies. To patch the hole, use sterile tape or a portion of a fresh aluminum seal.
Kits Shipped at Room Temperature
This type of kit from Addgene will arrive at room temperature with the glycerol stocks thawed. To access the plasmids, keep the plate stable and remove the cap strip carefully to avoid splatter and cross contamination. We suggest you inoculate the samples into fresh bacterial cultures and grow them overnight to make sure that all samples are growing. You can then add 50% glycerol and store the freshly grown samples at -80°C. Please note that storing the samples at -80°C immediately upon arrival may reduce survival in future plasmid recovery attempts.
After proper storage of your new Addgene kit 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.
Inoculate an Overnight Liquid Culture - using a liquid culture will allow you to grow enough bacteria for plasmid DNA purification.
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 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.
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 those Addgene has performed on the plasmid.
Please visit www.addgene.org and search the catalog number of your kit 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 kit number in future publications.
Thank you for requesting a kit from Addgene. If you have questions or would like to speak with an Addgene scientist, contact us at +1-617-225-9000 or email [email protected].