CQLS has installed a new Leica Stellaris 5 Confocal Microscope System which replaces an older confocal system. With this type of microscope one can obtain 3D sectioning of fluorescently labeled cells, or tissues for clearer, sharper images of specimens. People have used confocal systems to examine structures within living or fixed cells and to examine the dynamics of cellular processes.
3D rendering of veins in maize leaves. Yellow: Pin1a-YFP in cell membrane, Red: DR5-RFP in endoplasmic reticulum. Image courtesy of Camila Medina.
A confocal system has the capacity to image in Z and time to better visualize location in 3D than widefield fluorescence microscope by using a pinhole to eliminate out of focus light. The system has a white light laser (WLL) with tunable excitations from 485 nm up to 685 nm in addition to a 405 nm laser and sensitive HyD S detectors with a detection range from 410 to 850 nm. Additionally, the Stellaris system also has TauSense, a set of tools based on fluorescence lifetime information with potential to eliminate autofluorescence, and LIGHTNING which expands the extraction of image details for both classical imaging range and beyond the diffraction limit (120nm).
We will be offering free training and imaging time during this fiscal year to those people who have a project ready for imaging and in order to help with grant writing for future imaging projects. Contact Anne-Marie Girard to discuss potential projects or for more information about the system or its capacities.
Our BluePippin instrument is used for DNA size selection. Several genomic applications benefit from collecting only DNA fragments within a specified size range from a pool of DNA. This service is often used before Illumina sequencing to remove undesired PCR peaks or before long read sequencing to increase the proportion of fragments greater than a given length in a library.
Using pulsed-field electrophoresis on pre-cast gel cassettes, a DNA sample is separated and fragments within the target range are eluted into buffer. Each cassette can run up to 5 samples at once. Up to 5 ug of DNA can be loaded into each well.
The following cassettes are available at CQLS:
3% agarose, 100-250bp
2% agarose, 100-600bp
1.5% agarose, 250bp – 1.5kb
0.75% agarose, 1-50kb
The desired size range must lie within the total size range capability of the cassette. For example, between 200-350 bp on a 2% agarose cassette would be an acceptable range.
The expected collection yield of target sizes is approximately 50-80% based on product validation studies. Yield can vary greatly depending on the desired range and input fragment sizes.
Before and after size selection of Illumina sequencing library to remove undesired peak at approx. 300 bp
Contact Katie Carter for inquiries about this service.
Aaron Trippe discusses the changes and challenges of working with the PacBio Sequel since 2016. He discusses improvements in the technology since 2016 and has advice for user who would like to utilize this service.
Aaron Trippe, our long-time PacBio technician, stands next to the CGRB’s Pacific Biosciences Sequel.
Q1: How long have you been running the PacBio sequencing service at the CGRB?
The CGRB was one of the early adopters of the Sequel, the second phase of long read genomic sequencing technology from Pacific Biosciences. It arrived here on campus in August of 2016. Since then the technology has made significant improvements to the user-interface, and has tremendously increased read lengths and output.
Q2: You started up the PacBio sequencing service at the CGRB. What has been the most challenging aspect about developing this service?
Aside from the continually changing and evolving technology, one of the most challenging aspects of the service is getting everything you feed the machine to produce optimal results. One of the advantages of the technology is that you are sequencing native DNA, but that also makes it challenging when working with an organism that traditionally is difficult to work with and considered problematic. Finding ways to produce super clean and high molecular weight DNA from just about everything is probably the largest hurdle to working with the technology as a service provider. The keys to success are definitely within the sample quality. Having pure, high molecular weight DNA is essential to take advantage of the long read aspect of the technology, and is directly correlated to the quality of the sequencing output.
Q3: What type(s) of project(s) would you recommend to use PacBio’s long read technology?
The technology is great for just about any sequencing application. With the long reads, you have access to regions of DNA that were not previously accessible due to repetitive regions in genomic DNA. There is enough output to multiplex several microbial genomes on a single SMRT Cell. Complete sequences of multiplexed amplicons using Circular Consensus Sequencing for high fidelity reads of shorter inserts. With the read lengths exceeding that of RNA transcripts, Isoform sequencing using the Iso-seq application is also available for obtaining complete transcripts.
Q4: Favorite or most interesting project you’ve worked on?
Since managing the PacBio Sequel, I’ve gotten to work with plants, animals (vertebrates/invertebrates), fungi, bacteria, and insects for the local scientific community, and beyond. I can’t say that I have had a favorite organism, and they have all been interesting projects, but overcoming challenges with successful results always feels rewarding.
Note: We wish Aaron the best as he purses a new opportunity and are grateful he was able to develop a successful PacBio Service at the CGRB! For future sequencing inquires please contact Katie Carter.
