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TypeProtocol...resolution (the ability to distinguish between two discrete objects). Figure 1 depicts an image of a compound... sample information so that you or others can interpret your images in the future. Conclusion Like any...
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Isolating a Monoclonal Cell Population by Limiting Dilution
TypeProtocol...results in cell populations that are more likely to retain stable transgene expression. Other methods of generating...well in sparse/individual cultures due to lack of secreted growth factors, so not every cell type will be... -
Protocol - pLKO.1 – TRC Cloning Vector
TypeProtocol...position 15-19. Avoid targeting introns. Avoid stretches of 4 or more nucleotide repeats, especially repeated...Incubate for 20-30 minutes at room temperature. g. Retrieve HEK-293T cells from incubator. The cells should... -
Protocol - How to Design Primers
TypeProtocol...end, and the 5’ end of the primer usually has stretches of several nucleotides. Also, both of the 3’ ends... -
Plasmid Modification by Annealed Oligo Cloning (with Protocols)
TypeProtocol...cloning can be used anytime you need to add a short stretch of DNA to a plasmid, such as: Changing a Multiple... -
Ligation Independent Cloning
TypeProtocol...cloning scars"). A “stuffer” sequence allows for electrophoretic separation of linearized vector from the reaction... -
Protocols for Molecular Biology, Plasmid Cloning, and Viral Preps
TypeProtocol...Modification by Annealed Oligo Cloning Add a short stretch of DNA to a plasmid Gibson Assembly Combine overlapping... -
General Transfection
TypeProtocol...first. Pro-Tip The pH of this solution will drift pretty rapidly upon addition of acid or base. Add only... -
Protocol - How to Ligate Plasmid DNA
TypeProtocol...indicates the various controls: Control Ligase Interpretation Uncut vector - Checks viability of competent... -
Transfection for Recombinant Antibodies
TypeProtocol...dropwise. Cap the flasks and swirl 5–10 times to mix. Return the flask to the incubator. Section 3: BCD Feed... -
Protocol - How to Run an Agarose Gel
TypeProtocol...details on doing diagnostic digests and how to interpret them please see the Diagnostic Digest page. Purifying... -
Pouring LB Agar Plates
TypeProtocol...manufacturer to make sure this is true in your case. Retrieve your molten agar mix from the autoclave. Pro-Tip... -
Plasmid Cloning by PCR (with Protocols)
TypeProtocol...little chance for mis-priming, so you can use a pretty wide range of annealing temperatures, but you may... -
Coomassie Purity Stain of Recombinant Antibodies
TypeProtocol...background staining. Pro-Tip A large shift in the electrophoretic mobility of the bands (sample AR0016 in Figure... -
Technologies Enabled by NanoLuc® Luciferase
TypeBlog Post...powerful the FRET signal. In well-designed FRET-based biosensors, the intensity of the FRET signal can ...packaged into a retrovirus for infection of host cells. The fluorescent moiety of the BRET reporter was ...proteins together thereby increasing their FRET signal. FRET is a non-radiative transfer of energy from...accuators. The researchers took a FRET reporter and adapted it into a BRET reporter by using NanoLuc® Luciferase... allows researchers to convert FRET biosensors into more versatile BRET biosensors that can even be paired...PMC5171807. 6. Machleidt, T., et al. (2015) NanoBRET-A novel BRET platform for the analysis of protein-protein... on the Addgene Blog Learn more about FRET Tips for Using FRET in Your Experiments Learn more about Fluorescent... -
Using AAV for Neuronal Tracing
TypeBlog Post...developed a new AAV variant, rAAV2-retro, that migrates retrogradely at higher rates than any other AAV... AAV2-retro vectors with a variety of transgene/promoter combinations. Check out our retrograde AAV blog...Selective retrograde transsynaptic transfer of a protein, tetanus toxin, subsequent to its retrograde axonal...strengthened during memory formation and memory retrieval. In neuronal tracing experiments, brain tissue...bodies to the terminals of their axons; while retrograde tracing outlines neurons in the opposite direction...axons to their cell bodies. Anterograde and retrograde tracing take advantage of existing transport ...myosin, and enzymes for transmitter synthesis. Retrograde transport is used for transport of endocytosed... -
Viral Vectors 101: Types of viruses
TypeBlog Post...viruses: gamma-retrovirus, lentivirus, adenovirus, and adeno-associated virus. Gamma-retrovirus Retroviruses...type of gamma-retrovirus is Murine Leukemia virus (MLV). Naturally isolated gamma-retroviruses have been ...Lentivirus is a type of retrovirus and shares many common features with gamma-retrovirus including genome architecture... nucleus as gamma-retroviruses do. Fun fact: Since lentivirus and gamma-retrovirus are so closely related...expression (Robinson, 1982). To add insult to injury, retroviruses generally have moderate to high immunogenicity...immune response in the host. So why use a gamma-retrovirus in the lab? Well, they are essentially evolutionarily... types with a high efficiency. However, gamma-retrovirus infection is restricted to dividing cells only... -
Avoiding the Dark Side of Fluorescent Protein Fusions with mOX FPs
TypeBlog Post...compose the secretory pathway, endocytic vesicles, mitochondria, lysosomes or they are secreted into the ...blocked from proceeding through the secretory pathway, and retained in the ER. FPs have been undeniably...include the quality control of secretory proteins in the Endoplasmic Reticulum and live cell imaging approaches... the power of the Dark Side." --Darth Vader in "Return of the Jedi" While Vader was referring to the evil...what if an FP fusion with a resident endoplasmic reticulum (ER) protein instead localizes to the cytoplasm...position requirements. For example, a KDEL ER retrieval sequence only functions at the extreme C-terminus...residues form inappropriate disulfide bonds in the secretory pathway (Figure 1) [4,6–8]. Additionally, FPs ... -
Viral Vectors 101: Viral Vector Elements
TypeBlog Post.../gamma-retrovirus Lentiviruses are a class of retrovirus (so all lentiviruses are retroviruses, but not...not all retroviruses are lentiviruses.) This means that lentivirus and gamma-retrovirus share a lot of...as tools in the lab. We will discuss the gamma-retrovirus, lentivirus, adenovirus, and adeno-associated...the common components outlined above for gamma-retroviruses. While not required for lentiviral production... Fig. 1 - Viral vector components of retrovirus Adeno-associated virus AAV’s elements are...are quite different from gamma-retrovirus, but they perform very similar functions to infiltrate the host...in common with AAV, just like lenti and gamma-retroviruses have a lot in common with each other. Below ... -
Measuring Kinase Activity at the Single-Cell Level with Kinase Translocation Reporters (KTRs)
TypeBlog Post...monitoring live single-cell kinase activity is FRET, but FRET reporters are challenging to design and difficult...multiplex. Problems with FRET kinase reporters Difficult to Multiplex: FRET reporters are comprised of...be multiplexed together. Challenging to Design: FRET reporter activity depends on both the distance and...difficult to predict. Slower Dephosphorylation Rates: FRET-based reporters can have slower dephosphorylation... than KTRs which is hypothesized to be caused by FRET reporters having phosphorylated conformations that... activity. Advantages of KTRs When compared to FRET reporters for measuring kinase activity, KTRs have...KTRs also have some disadvantages when compared to FRET reporters. First, KTRs monitor global kinase activity...