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Choosing Your Fluorescent Proteins for Multi-Color Imaging

Posted by Guest Blogger on Oct 9, 2014 11:00:00 AM

This post was contributed by Kurt Thorn of the Nikon Imaging Center at UCSF.

A common requirement for live cell imaging experiments is the ability to follow multiple fluorescently tagged species simultaneously. To do so with fluorescent protein labels requires multiple fluorescent proteins whose excitation and emission spectra differ sufficiently for them to be imaged in distinct fluorescent channels on the microscope. With the proliferation of fluorescent proteins in recent years, there are many fluorescent protein combinations that can be imaged together, but this also means that the choice of fluorescent proteins requires some thought.

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Topics: Lab Tips, Fluorescent Proteins

ICYMI: Addgene’s Plasmids 101 eBook

Posted by Marcy Patrick on Oct 2, 2014 1:25:00 PM

We'd like to thank all of the contributing Addgenies that made this eBook possible: Melina Fan, Matthew Ferenc, Larissa Haliw, A. Max Juchheim, Caroline LaManna, Margo Monroe, Kendall Morgan, Jason Niehaus, Marcy Patrick, Lianna Swanson, Julian Taylor-Parker

We'd also like to thank our guest contributor: Gal Haimovich of greenfluorescentblog.org for helping us explain why things glow!

Addgene's Plasmids 101 eBook is here: Enjoy more time developing clever experiments and less time researching basic plasmid features – download the Addgene Plasmids 101 eBook!

Our goal was to create a one-stop reference guide for plasmids. We’ve combined our Plasmids 101 blog posts from the last year with some additional resources to create one downloadable PDF you can save to your desktop for easy reference. Highlights include our guide to fluorescent proteins, information about promoters and ORIs, and tips for naming your plasmids.  

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Topics: Plasmid Elements, Lab Tips, Plasmids 101

Plasmids 101: How to Verify Your Plasmid

Posted by Lianna Swanson on Aug 28, 2014 11:34:00 AM

Congratulations, you have a plasmid expressing your gene of interest (YGOI) and are ready to dive into your functional experiments! Whether you’ve cloned the plasmid yourself or obtained it from a colleague down the hall, it is always a good idea to take some time to confirm that you are working with the correct construct, and verify that the plasmid you received matches the expected sequence. Here at Addgene, we use NGS-based quality control to confirm the sequence of all the plasmids we distribute. This method is time-intensive, so we recommend two other methods for quick plasmid verification: Sanger sequencing and diagnostic restriction digest.

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Topics: Plasmid How To, Lab Tips, Plasmids 101

6 Tips for Analyzing and Troubleshooting Sanger Sequencing Results

Posted by Lianna Swanson on Jun 26, 2014 10:23:15 AM

This blog was originally published on BitesizeBio here.

As part of my job ensuring plasmid quality at Addgene, I analyze 50-100 sequencing reactions a week. So I have developed some good habits that I wanted to pass on to you to make sure you are getting the most out of the data you get back from your sequencing runs.

When you run a restriction digest on a gel you always include proper controls like uncut DNA and the proper ladder. These controls help you properly visualize your results.The most important of those is to always look closely at the trace file (or chromatogram) of the sequencing results you get back from your favorite sequencing facility.

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Topics: Plasmid How To, Inside Addgene, Lab Tips

Tips for Using BLAST to Verify Plasmids

Posted by Jason Niehaus on May 29, 2014 9:29:00 AM

This post was updated on Dec 4, 2017.

At Addgene, we continually use the Basic Local Alignment Search Tool (BLAST) provided by NCBI. BLAST helps us compare the sequencing results of the plasmids in our repository with known reference sequences, such as full plasmid sequences provided by the laboratories that deposit their plasmids with us or other entries in NCBI’s numerous databases.

As our repository has grown over the years (we now have over 60,000 plasmids!), the number of sequencing results we analyze as part of our quality control process has steadily grown. On a busy week, we may need to analyze more than 200 plasmids as part of our quality control process. Consequently our team has refined our use of the BLAST web browser interface to be as efficient as possible.

If you find yourself frequently on the BLAST website to verify plasmids or validating your new clones, try these tips to make the most of your time and sequence! You might also enjoy seeing how our quality control process has changed with the introduction of next generation sequencing! 

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Topics: Plasmid How To, Lab Tips

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