How to Deposit Your Plasmids with Addgene

Posted by Tyler Ford on Jan 5, 2016 10:30:00 AM


At Addgene, it is our mission to make it easy for you to share plasmids. To achieve this goal, we will archive any plasmids you've deposited with us and distribute them to scientists at academic and non profit institutions worldwide. What's more, depositing is free! When we travel to talk to scientists about how we can help them share plasmids, we get many questions about how the deposit process works. To make things a little easier for you, we've written this post as a step-by-step guide to the online deposit process.

To deposit plasmids online, you will need to create an account with Addgene before starting the process.

After you have logged in to your account, you can begin the deposit process by clicking “Start Deposit” in the Deposit Plasmids menu:

Start_Deposit.jpg

Enter Article Information

As you can see, you have three options when starting the deposit:

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1. You can “Add Plasmids from a Published Article”: This option allows you to quickly associate your plasmids with the original publication where you described them. Enter the PubMed ID, paper title, or authors in the PubMed search box and choose the appropriate publication. This publication will be listed at the top of the final plasmid pages and scientists will be instructed to cite this article when using these plasmids.

2. You can “Add Pre-Publication or Unpublished Plasmids”: You can also deposit plasmids that are unpublished or that are pending publication. Many labs have unpublished plasmids that are very useful to the research community. We can always associate the plasmids with a publication at a later date. Enter your own custom description of the plasmids in the "Enter Plasmid Description" box. To request that your plasmids be kept offline until the release of any pending publication, please include “HOLD” in your description.

3. Submit Plasmids Using Our Spreadsheet: The spreadsheet option is recommended for depositing 15 or more plasmids that are similar (e.g. gRNAs in the same backbone). Our spreadsheet contains columns that correspond to the plasmid data we need; you can copy and paste your information directly into the file. This file can be emailed to deposit@addgene.org along with your GenBank files, plasmid sequences, and/or maps.

Add Plasmids to Your Article

After you’ve selected your publication or entered your description, the next page will allow you to add plasmids to your article:

Add_Plasmids.jpg

On the article page, enter the name, type, and description of each plasmid associated with this deposit as explained below:

1. Name – we encourage the use of descriptive plasmid names and using the exact names listed in your publication. Aliases for your plasmid can be added on subsequent pages.

2. Type - please choose from one of the following descriptions for your plasmid:

  • Encodes One Insert:

A plasmid with one gene or protein coding sequence cloned into it. 

  • Encodes gRNA/shRNA

A plasmid with one gRNA or shRNA sequence cloned into it. gRNAs are used for directing CRISPR/Cas9 to the appropriate genomic location while shRNAs are used to knockdown expression of mRNAs with complementarity to the shRNA sequence.

  • Empty Backbone

A plasmid designed for cloning a gene/insert into it. Empty backbones often contain epitope tags or fusion proteins that are appended to new inserts through cloning.

  • Encodes Multiple Inserts

A plasmid encoding multiple inserts can contain multiple genes or small non-coding RNAs.

*Note - If you will be depositing multiple plasmids and would prefer to send us your plasmid information in spreadsheet format, e-mail us at  deposit@addgene.org

Exclamation.jpgSpecial Cases

Please contact us at deposit@addgene.org or +1 (617)-225-9000 if your plasmids fall into one of the special cases below:

 

  • Pooled Library

A pooled library is a set of plasmids all built with the same backbone and only differing in the gene/insert. Pooled libraries are normally supplied as liquid DNA with all of the plasmids in the library in a single tube. The number of unique plasmids in a pooled library can range from a few hundred to millions. Pooled libraries must be verified by next generation sequencing. Please contact us at deposit@addgene.org or +1 (617)-225-9000 if you’d like to distribute your pooled library through Addgene.

  • Bacterial Strain

Bacterial strains that you’ve created and that are necessary for use of a plasmid you are depositing can be distributed through Addgene. We will need information on how to verify any genomic alterations unique to your strain. Please contact us at deposit@addgene.org or +1 (617)-225-9000 if you’d like to distribute your bacterial strain(s) through Addgene.

3. Description – please indicate the gene cloned into your plasmid, any tags or fusion proteins contained in the plasmid, any mutations, and the intended purpose of the plasmid.

Once you've named and described all of the plasmids in your deposit, you should click the "Enter Data" button to begin entering the specifics of your plasmids.Enter_Data.jpg

Enter Plasmid Information

During the remainder of the process you will enter plasmid data in the pages described below.

If you will be depositing pooled libraries or bacterial strains, please contact us at +1 (617)-225-9000.

You can complete these pages sequentially by entering the appropriate data and pressing “Save and Continue to Next Step” (green button at the bottom of the screenshots below) or you can click the save button and jump around the different pages by clicking on the appropriate boxes at the top of the page.

Exclamation.jpgPro Tip!

Be sure to press the blue “Save” button at the bottom of the page before moving onto a different page so that any data you enter is not lost.

 

1. Sequences, Maps, and Files:

Please provide any full or partial plasmid sequences and any vector maps. The more sequence data available, the better. We highly encourage you to upload full sequence data whenever possible. Note that full plasmid sequence can be assembled or theoretical-it does not have to be entirely verified by sequencing.

Upload any support files that will make it easier for scientists to best utilize your plasmids (these can be protocols, GenBank files, etc).

Enter_Info.jpg

2. Gene and Insert

*Note - This page will not appear for empty backbones

Please enter the name of any gene contained within your insert as well as the total size of the insert. You can select the species that the gene or insert sequence came from and include the GenBank ID associated with it. Entering this information will populate a list of possible Entrez Gene matches to your insert. Select the appropriate gene if it appears in the Entrez Gene list as this will make it easier for other scientists to find information related to your plasmid.

Exclamation.jpgPro Tip!

To get the Entrez Gene list to auto-populate with the correct gene, entering the full, official NCBI gene name in the Gene/Insert box works best. Once you have selected the appropriate gene from the list, save your progress and you can then modify the gene/insert and alternative name boxes as needed.

Use the “Relevant mutations/deletions” box to describe any mutations within your insert using standard mutation notation. Please indicate the amino acid change as well as any functional consequences of that change. For example, “Mutated aspartate 123 to Lysine (D123K), lowers biosensor affinity for calcium.”

Please describe any tags or fusions (eg. His, FLAG, EGFP, etc) on your insert. Use the drop down menu to indicate whether the tag is on the N- or C – terminus and whether it was present in the backbone or was a part of the insert (contained within restriction sites used to clone the insert into the vector backbone).

As part of our quality control process, we will sequence features you annotate on this page.

Gene_Insert.jpg

3. Cloning Information:

Please enter the name of the vector backbone you cloned your insert into and fill out the appropriate boxes with the vector backbone manufacturer, vector backbone size, total size of your vector + insert, any modifications you made to the backbone, and the vector type.

Exclamation.jpgPro Tip!

Reference the Addgene ID in the “backbone manufacturer” box for backbones requested through Addgene.

 

The vector type should describe how your plasmid is intended to be used. For instance, if your plasmid is designed to allow you to produce large amounts of your insert in E. coli, then this is a Bacterial Expression vector. If your plasmid can be classified in multiple categories, please check all of the appropriate categories (eg. for a lentiviral vector to be used in mammalian cells, check Lentiviral and Mammalian Expression). This information will make it easier for scientists to find your plasmid by type.

Cloning_Info.jpg

If your plasmid has an insert, you will be prompted to indicate how you cloned the insert – the cloning method used, restriction enzymes used (if applicable), and primers that can be used to sequence over your insert (input the primer name and sequence directly into the box as shown below). This information will greatly facilitate Addgene’s quality control processes and will make it easier for scientists to use your plasmid.

Input cloning information to describe how your gene was inserted in the plasmid backbone and what primers can be used to verify it as shown below:

Primer.jpg

4. Growth and Distribution:

Please indicate how Addgene and other scientists can propagate your plasmid. Accurate information will greatly facilitate the deposit process and allow other researchers to access and use your plasmids sooner.­

Whenever possible we propagate plasmids in the standard cloning strain DH5α. Please indicate if your plasmid cannot be propagated in DH5α. For plasmids with highly repetitive sequences (which can be prone to recombination in bacteria) such as lentiviral, retroviral, and AAV plasmids, we recommend the NEB Stable strain. For plasmids containing the ccdB gene, such as Gateway vectors, we recommend the ccdB Survival strain.

Growth_Distribution.jpg

5. Verification

On this page you will see a preview of the final plasmid page that will appear on Addgene’s website once your plasmids are available online. Please review all of the information on this page to make sure it is correct. You can easily update the information by navigating to the previous pages using the links at the top of the page.

Verification.jpg

Once you are satisfied with the plasmid information, click on the green “Save Verified Data and Return to Article” button to return to the table listing all of the plasmids for this deposit. You can then enter the information for the next plasmids in your deposit.

Exclamation.jpgPro Tip!

If you have multiple plasmids with the same gene or vector backbone, you can copy some information from a previous plasmid in your deposit by selecting the plasmid to copy from the drop down menu and clicking the blue “Copy” button.

Note* – The “Copy” button will only appear after you have entered information for your first plasmid and can be used on each individual page (Gene and insert, Cloning Information, Growth and Distribution).

Copy.jpg

Once you have completed the data entry process for all of the plasmids in your depost, return to the Add Plasmids page and click the green “I am done entering data. Request Deposit Kit” button.

Request_Kit.jpg

Clicking this button will allow you to choose which plasmids you wish to deposit, associate the plasmids with a Principal Investigator (PI), provide us with your shipping address for deposit materials, and agree to our terms and conditions. 

Once this process is complete, you will receive a Deposit Confirmation email from us with a unique Deposit ID. Addgene will send you a package containing instructions on how to prepare your plasmids, and pre-paid shipping materials you can use to send us your plasmids.

When your plasmids arrive at Addgene, we will notify you that we have received them and they will undergo our quality control process to verify key features of your plasmids. Our friendly Addgene scientists will notify you once your plasmids are online and available to the research community.


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