...cells are passed through the path of a laser and interrogated with various visible and fluorescent light sources...path of the laser in single-file where it is interrogated by the optical system (Figure 1).   ...a variety of fluorescent light sources. When interrogated with a fluorescent light source, a cell expressing...expression, the further the shift of a cell from the negative control, the higher the level of expression. A...  Protein expression, measured using antibody conjugated fluorophores Fluorescent antibodies get around...staining, the primary antibody against a target is conjugated to a fluorochrome. In indirect staining a primary...antibody binds to a target and a fluorochrome-conjugated secondary antibody binds to the primary antibody...

... present in the gate.   Figure 1: Example of yes-no flow plot of the (negative) control (left)...MFI, you can still continue gating populations of interest with normal gates, just based on a histogram...way, the GFP- population should overlap with the negative control population (Figure 1). Based on where ...where the GFP- population ends, you can draw a gate which selects anything to the right of it, marking it ...left) or with the sample (right). Negative and positive samples are clearly distinguishable as two separate...treatment processes, you may see a shift in the negative population, so it no longer overlaps with the ...from Figure 2 that determining where to place your gate based on the dot plot would be complicated.  ...

...methods such as Golden Gate or Gibson assembly. Golden Gate Assembly of gRNAs Golden Gate Assembly uses Type...needed for the final ligation step. Plasmids are then digested using BsmBI and ligated into Cas9 or dCas9...lab. Scaling up your multiplexed gRNAs with Golden Gate and Gibson assembly methods If you want to scale...destination vector. The first step in CRISPR/Cas9 Golden Gate multiplexing is to clone the oligonucleotides specifying...the procedure will vary slightly.   Two Golden Gate options that are available from Addgene follow these...repression), and Fok1-dCas9 (dimeric nuclease). Gateway assembly method Frew Lab Multiple Lentiviral Expression...should be compatible with type IIS enzyme BfuAI. Gateway cloning is then used to combine the multiple gRNAs...

...RepB) necessary for replication in various hosts (Pagatto and Dillon 2001).  The plasmid has its own replication...self-transmissible, meaning that the plasmid is conjugative and carries the genes necessary for transfer ...with Shown to work in IncN pCU1 Gram-negative  Agrobacterium spp., Bradyrhizobium spp., Rhizobium...Rhizobium spp. IncP RK2 Gram-negative  Achromobacter parvulus, Acinetobacter spp., Aeromonas...campestris IncQ RSF1010, R300B, R1162 Gram-negative and Gram-positive  Acinetobacter calcoaceticus...campestris, X. maltophilia IncW pSa, pR388 Gram-negative  Acinetobacter calcoaceticus, Aeromonas liquefaciens...Zymomonas mobilis pBBR1 based pBBR1  Gram-negative  Alcaligenes eutrophus, Bartonella bacilliformis...

...in combination of two conjugated single fluorophores, like APC-Cy7. Conjugating Cy7 to APC results in ...antibody is conjugated to. Since CD45 is a common marker, finding an antibody already conjugated to my fluorophore...Harvard Medical School. His projects include investigating negative regulators of T cells and cytokine signaling...proteins is through treating cells with fluorophore-conjugated antibodies that attach to a specific target. ...whether that fluorophore appears alone or in conjugation with another fluorophore. The brighter a fluorophore...fluorophore when excited by the red laser. When conjugated to Cy7, however, (APC-Cy7) the brightness changes...fluorescent reporters, such as GFP, and fluorophore-conjugated antibodies together in the same color panel. ...

...buffer components are compatible with your conjugate and conjugation chemistry.  Quantification Once you’ve...antibodies to their parental hybridomas. Aggregation and storage Aggregation of antibodies may impact their quality... used for recombinant antibody production is Komagataella phaffii (a.k.a  Pichia pastoris) because it ...concentration using these columns. If you are conjugating your antibody in a downstream step, you’ll want...quality and efficacy (Wang et. al., 2018). Aggregation can arise from a shift in temperature, pH, or salt...concentration (Amin et. al., 2014). Antibodies may even aggregate just from dropping the tube they are contained... PMID: 25819219. Wang W, Roberts CJ. Protein aggregation - Mechanisms, detection, and control. Int J Pharm...

...not the other. 3) The beads are collected by centrifugation or a magnet. 4) Unbound proteins are washed...Protein G, or the recombinant Protein A/G are conjugated to agarose or magnetic beads and allowed to bind...target protein. The beads can be collected by centrifugation (agarose beads) or a magnet (magnetic beads... cases like these, antibodies can be directly conjugated to agarose or magnetic beads with commercially...commercially available chemical agents. Directly conjugating the primary antibody has the additional benefit of...provides a large area for binding, but antibodies conjugated within the pores are often inaccessible to their... be much smaller than agarose beads. Proteins conjugate on the surface of magnetic beads, and are therefore...

... derivative. For propagating plasmids expressing the ccdB gene (important in Gateway cloning). F-mcrA ... derivative. For propagating plasmids expressing the ccdB gene (important in Gateway cloning). F- gyrA462...Chloramphenicol High competency cloning and propagation of large plasmids, ligated DNA, and libraries, nalidixic acid... and determine whether it is appropriate for propogating your plasmid or carrying out your experiment....experiment. History of E. coli strains E. coli are gram-negative, rod shaped bacteria that were named after Dr....Consequence dam DNA adenine methylase mutation (GATC) Preparing unmethylated DNA, important when trying...plasmid, encodes proteins needed for bacterial conjugation. Genes listed on F´ are wild-type unless indicated...

...antibodies must be either sourced already conjugated or conjugated in the lab. In the indirect approach, ...idea of the basics of immunoimaging. A scientist conjugates an antibody with a signaling molecule, the antibody... indirect approach, the signaling molecule is conjugated to the secondary antibody instead of the primary...of interest is found in low amounts or if the conjugated signaling molecule has a weak signal. For flow... are easy to source and are readily available conjugated to a number of different signaling molecules...., you can simply keep a stock of conjugated secondary antibodies on hand and use them to easily test different...sample. Incubate the sample with the primary (unconjugated) antibody. Perform a series of wash steps to...

...Ebola virus belongs to the group of nonsegmented negative sense RNA viruses, along with other viruses that...minigenome?   The EBOV genome is a nonsegmented, negative sense RNA strand approximately 19 kb in length...systems have been developed for many nonsegmented negative sense RNA viruses (reviewed in 2, 3). Other important...translated) by cellular machinery. NP encapsidates the negative sense, single-stranded RNA minigenome. The viral...polymerase to synthesize additional copies of the negative sense minigenome, thus modeling genome replication...transcription of the reporter gene. The polymerase will elongate the mRNA until it reaches the gene end signal,...precautions, these systems allow researchers to investigate viral processes in extreme detail. The mechanism...

...experiment-based investigation of a hypothesis: proper controls! For every experiment, an investigator needs a ...empirically.  Negative controls: untreated cells, empty vector control, and non-targeting control Negative control...plasmid-based experiments employ transfection, negative, positive, and replicate controls.  Transfection...mentioned above) could also serve as an important negative control. In this context, the empty vector control...your observed result. Non-targeting controls are negative controls that produce a similar product, but do...be highly specific to the experiment and the interrogation of the independent variable. Replicate controls...experiment, incorporating additional positive and negative controls, performing multiple replicates, and ...

...pCAGGS-TAL-NC2 Charles Gersbach pcDNA3.1-GoldenGate, pcDNA3.1-GoldenGate-VP64 Maria-Elena Torres-Padilla pTALYM3...74334 pCR8-Magneto2.0-p2A-mCherry Gateway 74333 pCR8-Magneto2.0 Gateway 74307 pAAV-CMV-DIO-Magneto2.0...PRESTO-TANGO kit which allows researchers to interrogate the druggable human GPCR-ome using a quick, affordable...destination vectors using low-cost, one-pot golden gate cloning that results in the creation of many E. ...The collection is compatible with both standard Gateway cloning and the FNLCR Combinatorial Cloning Platform...Red recombinase enzymes, as well as a dominant-negative mutator allele of the E. coli MMR protein MutL...Ekker’s lab, is a modified version of the Golden Gate TALEN kit (GGT) that streamlines the assembly of...

...on the Multisite Gateway® technology from Life Technologies. The MultiSite Gateway Kit is a customizable... systems. The MultiSite Gateway Kit contains a set of 12 promoter-less Gateway® entry clones to be used... phage integrase system which is the basis for Gateway cloning. The combination of all 11 att pairs in...site and a linker region. The PCR product is then ligated into the MCS/SacI site in pUCXKT, removing the ...customizable Gateway®-based toolkit that allows scientists to generate fusion proteins from any ORF available...as a standard entry clone. ORFs from an ORFeome Gateway library or any PCR-amplified ORF cloned into pDONR221...heterologous promoter outside of the Multisite Gateway® cassette, you can specifically express your new...

...valuable when first optimizing conjugation in strains that don’t conjugate that well. This is because in...exchange. Here, a mutated allele introduced from a conjugative plasmid replaces the chromosomal copy. This is...bacteriophage lambda pir protein. This allows conjugation between a pir-containing donor and the recipient...inspired by this work and sought to develop these negative selection markers for allelic exchange. To do ... which we call “pTOX” in a broad group of Gram-negative Enterobacteriaceae including in E. coli O157:H7... to facilitate modification of strains where conjugation may be difficult, we have also included versions...in these circumstances, the conjugation can be so inefficient, that instead of the resulting colonies ...

...MiniPromoters MiniPromoter Source Gene Construct Reporter Negative Control N/A pEMS1312 cre N/A pEMS1301 cre/EGFP...

...than the negative control, and a weak signal has a longer exposure time closer to the negative control....specificity and exposure time. We first have a negative control which does not have primary and only contains...autofluorescence i.e., background noise. We then compare the negative control to a sample with the primary included ...background noise. If the sample images similarly to the negative control, that informs us that the primary antibody...non-specific binding. If the sample differs from the negative control, such as showing a very bright signal ...exposure time of the primary antibody compared to negative control also determines the signal’s strength....comparing your sample to. For example, we use our negative control to compare exposure times of our samples...

...overwhelmed if you don’t delegate - as I quickly learned! I was a poor delegator at first. I knew what ...team - which is every manager’s dream. Learn to delegate effectively  As a manager your role is not to ...more work (this is a big hint that you are not delegating enough!). There is no way around the fact that...possible so I could more easily identify what to delegate and to whom (make sure that person has time for...available, and encourage questions. Unless you’re delegating a recurring task to a seasoned report, there ...there should be questions! Remember that delegating is not just a way to create more time for yourself, but...need to check myself sometimes to make sure I am delegating well, but it does get easier over time! Communicate...

... the negative selection marker, but cells with random recombination may incorporate the negative selection...One of the most powerful strategies to investigate a gene's function is to inactivate, or "knockout",...resistance gene (NeoR) flanked by homology arms. The negative selection marker HSV-tk is used to select against...construct has randomly integrated into the genome, a negative selection marker like the HSV thymidine kinase...resistance gene and then a counter selection for the negative selection marker - this latter step will kill ...thus disrupted, or knocked out. Use positive and negative selection to find correctly recombined cells. ...selection marker selects for recombination, the negative selection marker selects against improper, random...

...introduce Mobile-CRISPRi by bacterial conjugation For gram negative bacteria, Mobile-CRISPRi is introduced...Mobile-CRISPRi stably integrates into the genome and is propagated during normal DNA replication and cell division...introduced using the bacterial integrative and conjugative elements in Bacillus subtilis (ICEBs1) system...system. ICEBs1 carries the genes needed for conjugation, but can also carry cargo genes, like Mobile-CRISPRi...bacterium of interest following IPTG induction of conjugation genes and bi-parental mating. Following excision...genome, the Mobile-CRISPRi ICE element forms a conjugative plasmid-like DNA circle and is transferred to...et al. "Biology of ICEBs1, an integrative and conjugative element in Bacillus subtilis." Plasmid 86 (2016...

...pSC101 through a negative feedback cycle. As with DnaA, high concentrations of RepA negatively regulate its... require the host's replication machinery for elongation and termination. These types of plasmids tend...is carefully regulated by a mix of positive and negative regulators. The genome of E. coli is about 4.6...opposite directions away from the ori. In the elongation phase, a DNA polymerase synthesizes a new DNA... best tools to regulate DNA replication. DnaA negatively regulates its own expression, so high levels ...copy number for a given plasmid has been reached, negative feedback circuits reduce replication of the plasmid...proteins. In nature, such plasmids ensure their propagation by limiting their copy number and compensating...