...NGS assembles a larger sequence from many smaller sequences, regions with repeats may not assemble correctly...circular assembly. Most of the time, these plasmids are returned as one or two partial assemblies. If we...complete. After the run completes, we begin our assembly process. Again, thanks to our partners at seqWell...from each sample in our pool and individually assembles the reads into a single FASTA sequence that our...that are particularly difficult to sequence and assemble, including GC rich regions. For example, the CAG...

...toehold switches for Zika detection We and other members of the lab had been working on advancing the paper-based...Aedes mosquito (Messina et al, 2016). We quickly assembled a team of students, postdocs and professors to...also were careful to set up clear tasks for each member of the team to accomplish so that many elements...Sensor Constructs at Addgene As we report, we have embedded this next generation of toehold switch-based RNA...further develop our rapid prototyping and sensor assembly pipeline for future emerging pathogens or health...synthetic biology, nanomaterials science, and self-assembly for applications in health, energy, and sustainability...Listen to Our Interview with Previous Collin's Lab Member and Sample 6 Cofounder Michael Koeris Learn How...

...experiments: Retrieve Ensembl Transcript ID(s) of your favourite gene(s) from Ensembl genome browser: https...https://www.ensembl.org/index.html Design crRNA/ssODN: Using Ensembl Transcript ID(s) as an input query...reagents, we scaled up these tagging experiments by assembling csRNPs in a 96-well plate format and tested parallel...reagents for the knock-in experiment were pre-assembled in a 96-well plate. Glioma stem cells were transfected...be found at Desktop Genetics .For ssODN design, remember to incorporate PAM-blocking mutations (e.g. convert...in bulk and stored in small aliquots at -80 oC. Assemble csRNP: Anneal crRNA and tracrRNA using a PCR block...

...permeabilize the cell membrane to allow antibodies to pass through the cell membrane. Formaldehyde fixation...degree of internal complexity such as extensive membranes produces a greater SSC.  Protein expression, measured...targets, measured by fixing and permeabilizing membranes For intracellular protein targets, users must ... dissolving lipids including those of the cell membrane. In some cases, this can mask epitopes such that...propidium iodide, can only enter cells with disrupted membranes as is the case with dead or dying cells. DNA binding...permeabilization as these methods require damaging the cell membrane and will cause all cells to stain positive. If... amine reactive dyes can only pass through the membrane of dead cells. Once in the cells, amine reactive...

...and plasma membrane reporters as well as secretory proteins or proteins targeted to membrane sub-domains...cleavage upon co-expression and spontaneous self-assembly. In an effort to obtain even more precise temporal...individual reporter constructs or as the fully assembled pMemoryArray.   Yang et al., Nat Methods 2014... Chem Biol. 2012 Apr 20;19(4):507-17. 10.1016/j.chembiol.2012.02.006. 2 Guntas et al., Proc Natl Acad...arrival in their target compartment (e.g. the plasma membrane, the extracellular space, or the lysosomes) can...plasmids with MXS-chaining. Analogous to modular assembly methods, such as BioBricks, the MXS-chaining method... organelles, a cell cycle indicator for mouse embryonic stem cells and a series of inducible promoter ...

...precursor protein. The EMBO Journal, 16(16), 5113–5122. https://doi.org/10.1093/EMBOJ/16.16.5113. PubMed ...commonly called retrovirus) family — of which HIV is a member — may seem like an unlikely candidate to use as...Figure 1 to compare the genomes of these two family members.   Figure 1: Gamma-retrovirus genome (left... RT-deficient genome. In both cases, the lipid membrane is the only component that is not dependent on...genes. It is derived from the host/producer cell membrane, and acquired as an envelope during exocytotic...E., & Teich, N. (1971). Noninfectious ARK mouse embryo cell lines in which each cell has the capacity ...

... a water bath with individual slots for tubes, remember that when you add the tube to the slots, it can...

...Cloning Design primers for Gibson Assembly and NEB HiFi DNA Assembly  OligoAnalyzer Tool from IDT: For... vector database: Find sequences for plasmids assembled from publications and commercially available sources...other primer characteristics. NEB Data-optimized Assembly Design (DAD): This tool designs overhang sets ...methods, such as restriction enzyme cloning, Gibson assembly, or In-Fusion cloning.  NEB Ligation Calculator...Beth Kenkel. Additional resources were added in September 2023 by Rachel Leeson.  Additional resources...

...following T, so that there are no gaps in the final assembly. In this case, 15 bp of homologous sequence is...

...your lab’s instrument before use. Last Update: September 2022 Equipment Centrifuge Microfuge tubes or other...

...our plasmid assembly pipeline. To date, this has resulted in high quality NGS-based assemblies for over ...sequencing depth and coverage in order to, for example, assemble hundreds of different plasmids from a single Illumina...amount. plexWell™ + Addgene = high quality NGS-assembled plasmids Because of the enhanced normalization...libraries is well suited to large-scale de novo assemblies of short constructs like plasmids. The plexWell...bioinformatics pipeline that will return high-quality assembled and circularized plasmids from plexWell™ NGS data...

... calcium chloride which permeabilizes the cell membrane so the bacteria can easily uptake your plasmid...an electrical charge to cells, to increase cell membrane permeability and thus transformation efficiency...example are bacteriophages that attach to bacterial membranes and inject their genetic material into the cell... more easily cross the negatively charged cell membrane. Physical methods such as electroporation or microinjection...microinjection actually pokes holes in the cell membrane so DNA can be introduced directly into the cell...electrical pulses to create transient pores in the cell membrane that genetic material can pass through. If you...

...and numerous other reasons. It is important to remember that we, scientists and non-scientists alike, ...spoke in support of science - he's one of the few members of Congress who holds an engineering degree. It...the postdoctoral engagement committee and active member of the university's Postdoctoral Association.  ...career that could simply cease to exist. But then November hit and panic ensued as “alternative facts” became...encourages open discourse with our fellow community members. The March for Science was an important first step...available to people who want to join the dialog and 2) remember that science should not be partisan: everyone ...science communicator, UBC Public Scholar, and a member of the Vancouver SciCATs. Follow her on twitter...

...they cannot cross cell membranes, so they are best used either with plasma membrane proteins (such as GPCRs...fluorescence wavelengths for FRET experiments. However, remember that, if you make an FP fusion, you are inserting... Most importantly, these compounds cross cell membranes, thus enabling FRET measurements in intact cells...Intact Cells Cy3NTAc Poly His  44 35-63 Plasma membrane protein, permeabilized cells  Cy5NTAc 32 25-...Intact Cells Cy3NTAc Poly His  63 50-90 Plasma membrane protein, permeabilized cells  Cy5NTAc 43 34-...Intact Cells Cy3NTAc Poly His  65 52-94 Plasma membrane protein, permeabilized cells  Cy5NTAc 59 47-...

...freely-available DNA-editing software packages to assemble your expected full plasmid sequences (examples...computer or server. This will ensure that future members of your lab can access and use the materials they...during the cloning, sample handling, or sequence assembly steps. All of our incoming plasmids are sequenced...include cloning junctions (where most sequence assembly errors occur), deletions, mutations, in-frame ...the 5’ and 3’ ends to verify that the sequence assembly and identity of the insert are correct. In other...Primer3 to help you design your custom primers. Remember, when designing a sequencing primer, you need ...

...superfamily is comprised of 48 human and 49 murine members. NRs permit the integration and communication of...of endocrine-hormone signaling. The success of members of this group stems in part from their ability ...xenobiotic lipids derived from the diet. Additional members of this family are called orphan receptors because...coregulators exist as multiprotein complexes, a member of a single coactivator complex can serve as a ...status of SRC-3 defines its association with other members of the complex, such as p300 and CBP histone acetyltransferases...multiple cell signaling systems. Activation of membrane receptors and signaling cascades conceivably allows...

... post: Epigenetic engineering with single-pot assembly Tracking cell-cell interactions New auxin inducible...service Epigenetic engineering with single-pot assembly By Eric Perkins Though it’s easy enough to contemplate...describes an elegant system that allows for the assembly of multiple DNA fragments efficiently and accurately... a “single pot.’   Figure 1: Single-Pot Assembly: a) Graphic depiction of chromatin within a nucleus...enzymes and reader-effector proteins. b) Golden Gate assembly (backbones available at Addgene) with examples...purposes, but the key component of the editing assemblies are sensor-actuators, either transcriptional ...

.... Since makerspaces are generally funded on a membership model - everyone contributes each month - with..., unlike your traditional biology lab, we have members with a wealth of skills to help do science on the...new replacement was not possible, but one of our members took an Arduino microcontroller and a motor controller...too expensive to make it practical - but we have members looking at how to re-use it as a platform for monitoring...fast, but with a wide range of skills among our members we’ve managed to learn a lot and cobble together... Victoria’s first hackerspace which now offers members and the public cheap access to personal manufacturing...

...kinetics over 14 hours of expression in C. elegans embryos. However, this initial timer was a tetramer with...mammalian cells, in C. elegans, and in Xenopus embryos. Of course, timers may be sensitive to changes ... of vesicle fusion and release from the plasma membrane by fusing various vesicle cargoes to FP timer ...controlling pancreatic differentiation) in mouse embryos (Miyatsuka et al.) Distinguish between models for...fusing Medium-FT to LAMP-2A, a lysosome-associated membrane protein, and following its subcellular localization...with changes in gene expression. In a developing embryo, for example, a researcher could determine whether...

... There is no way around the fact that new team members need to be trained, which may seem like a waste... a seasoned report, there should be questions! Remember that delegating is not just a way to create more...for yourself, but it will help develop your team members by giving them new opportunities. I still need...comfortable with awkward silences but I’ve learned to embrace them after one ended with the person in front of...going to get you what you really want to know. Remember that one on one discussion can be confidential...Protect your team Make you team feel safe. Your team members need to trust you and you need to encourage them...