...alternatives to the standard restriction/ligation, Gateway, or Golden Gate cloning methods. Whether it is for...based technologies We've described Sequence and Ligation Independent Cloning (SLIC) in a previous Plasmids...polymerase extension cloning) and SLiCE (Seamless Ligation Cloning Extract) to name two more. Likewise, there... work by significantly different methods. Golden Gate cloning does result in the seamless joining of fragments...location and NOT in the middle of your insert. Gateway cloning utilizes λ integrase to catalyze directional...

...process consisting of digestion, purification, ligation, and transformation. While restriction cloning... developed. Some of these include, TA cloning, ligation independent cloning, TOPO cloning, one step cloning...developed by Li, et al., in 2011. This method is ligation independent, it does not require purification ...Amplification + + Digestion + + Gel Purification + - Ligation + - Transformation + + The FastCloning process... reactions. These sticky ends are annealed and ligated in vivo. Detailed FastCloning procedure To start...highly simplified, purification-free, sequence-and ligation-independent PCR cloning method." BMC biotechnology...

...fluorescently-conjugated streptavidin to get more fluorophores per antibody, or conjugate with an enzymatic...primary antibody to bind the target, wash, add dye-conjugated secondary antibody that recognizes the primary...do simultaneous or sequential labeling is what conjugated secondary antibodies you have available. If the...only secondary antibodies you have on hand are conjugated with the same dye (say, you have Goat Anti-Rabbit... direct IF, using primary antibodies that are conjugated to fluorophores. However, the primary-secondary...or scFvs. Many, many options for fluorescently conjugated secondary antibodies are commercially available...

...Positive and negative controls Most biological experiments require positive and negative controls to ensure...experiments will benefit from positive (+) and negative (-) controls in addition to experimental samples...In this case, mouse samples from positive (+), negative (-) and experimental (?) groups were run on a ... check for tau molecular weight shifts. A negative control is a sample or group that is not subjected...experimental condition. In the example above, a negative control could be a third group of mice that is...receive either protein kinase inhibitor. Since the negative control group does not receive the drug, any response...

...dye-nitrilotriacetic acid (NTA) conjugates (Kapanidis, Ebright, & Ebright, 2001). These Cy/NTA conjugates work the same ...tag from 6 to 10 residues. While these Cy/NTA conjugates are not commercially available, they are easily...in intact cells. The Gradia Lab has deposited a gateway vector with Addgene, where you can insert your ...adjacent to each other on the same protein and a negative control experiment conducted on donor-only labeled... (Ni2+: nitrilotriacetic acid) n-fluorochrome conjugates." Journal of the American Chemical Society 123.48...Your Experiments Beware of Fluorescent Protein Aggregation Brush up on Your Fluorescent Protein Basics ...

...gRNAs. As each ligation reaction uses a pool of gRNAs as starting material, the ligation products are a... et al., 2014) individual genes for studies interrogating their functions. The continual advancements ...with straightforward restriction digestion and ligation steps to build barcoded lentiviral plasmids containing...lentiviral plasmids. Multiple rounds of digestion and ligation result in plasmid libraries containing combinations... developing and applying new technologies to interrogate and understand complex biological systems. He...

...though most MoClo reactions use Golden Gate cloning, not all Golden Gate cloning is MoClo. That’s because MoClo...Golden Gate Assembly with NEB’s DAD Interview with Nicola Patron on MoClo Plasmids 101: Golden Gate Cloning...hierarchy. MoClo originally developed from Golden Gate Cloning, in which a type IIS restriction enzyme ...MoClo that relies on Gibson and other non-Golden Gate cloning methods. MoClo’s organization schema MoClo...

...transduction (viral infection by bacteriophages), or conjugation (direct transfer from one cell to another). Bacteria...need special fertility genes to be capable of conjugation, and these are encoded by certain kinds of plasmids...to a recipient through direct contact. That’s conjugation! F factors can also be integrated into the bacterial...also have more than one function: for example, Gateway cloning relies on genes found on an E. coli fertility...contain this F plasmid is critical for successful Gateway cloning. In addition, scientists have engineered...101: Transformation, Transduction, Bacterial Conjugation, and Transfection Resources on Addgene.org Addgene's...

...it allows for better resolution of positive and negative populations, Careful panel design In both conventional... your fully stained samples. If you are using conjugated dyes, it is critical that your controls and your...variation. Tandem dyes (two fluorescent molecules conjugated together) can be especially sensitive to batch...batch effect. For example, when a PE molecule is conjugated with a Cy7 molecule, there is no telling exactly... (positive population) or won’t bind anything (negative population). It is strongly recommended that when...AntibodiesAntibodies 101: Buffers, Storage, and Conjugates Resources on Addgene.org Addgene's Antibody GuideAddgene's...

...to dimerization (i.e. EGFP) [1, 2] or even form obligate tetramers (i.e. DsRed) [3]. This is a problem ...localization, dynamics, and behavior of a POI. As an investigator, you want a fusion tag to be inert, to not produce...has gone into making FPs monomeric, yet many investigators remain ignorant of FP dimerization. Equally ...and difficult to ignore [1, 2]. 2. Fusion to an obligate dimer or oligomer Several cell proteins normally... is sedimentation equilibrium analytical ultracentrifugation [2]. Some affinity assays used in the papers...moderately dimerizing FPs such as EGFP, but not for obligate oligomers such as DsRed or high affinity FPs such...

...incomplete lysing/insoluble proteins, and protein aggregation. Protein degradation, which would result in low...from their binding. If you’re seeing protein aggregation, you may want to consider changing your incubation...incubation temperature. Some proteins aggregate at 95 °C, a common lysis temperature, so consider a longer...incubation (30–60 minutes) at 37 °C, if you suspect aggregation in your prep. If your proteins are the wrong ...binding site, allowing you to visualize them with a conjugated antibody. If you do not need such precise measurements...of alcohol. The increased SDS will increase the negative charge on the protein, allowing it to move more...Blot Doctor page, or robust discussions, like ResearchGate’s western blot forum.  Blocking Don’t skimp on...

...Plasmids from the Tsuyoshi Nakagawa lab are binary gateway vectors and can be used for promoter swapping and... al., 2007). Alternatively, if you prefer Golden Gate assembly, there are a series of plasmids from the...transformant with a single insertion because it will segregate 3:1 in the T2 generation. You also have no control...genotype in T1 than T2. Your transgene should not segregate in T1, but it probably will in T2. Laura Lee...Dinneny, J. R. (2013) ‘A robust family of Golden Gate Agrobacterium vectors for plant synthetic biology...PMC4426443. 4. Nakagawa, T. et al. (2007) ‘Improved Gateway Binary Vectors: High-Performance Vectors for Creation...

...a clear separation between positive (blue) and negative (gray) droplets. The no template control wells...The ddPCR software uses the ratio of positive to negative droplets is used to calculate the concentration...getting a clean separation between positive and negative droplets with these parameters, here are a few...leading to greater separation of your positive and negative droplets. No more than 50 cycles is recommended...but you can go as low as 1C/s. Increasing the elongation time to 2 minutes and the denaturation time to... Witte, Anna Kristina, et al. "A systematic investigation of parameters influencing droplet rain in the...

...of the first BiFC studies used the method to interrogate the subcellular localization of interactions ... may also work. You will also need to generate negative control constructs — more on this later. For more...fluorescence intensity of your BiFC constructs to your negative control to determine whether your PIPs actually...actually do interact. Choosing the right negative control is critical to the success of BiFC experiments. The... The FP fragments themselves cannot be used as negative controls because their expression level, stability...BiFC competition analysis can be used in lieu of negative controls (Kerppola, 2006). Here, one of the endogenous...

...the cytoplasm. That is, unfortunately, some investigators realize too late that they have missed the impact...confound quantitative imaging experiments or even negatively impact cells (see Figure 1). When behaving properly... properly, FPs enable researchers to investigate the localization and dynamics of fluorescent protein ...environment where it will localize [4]. Most investigators are surprised to learn that FPs are often poorly...overestimation of fusion protein activity. We encourage investigators to characterize FP-fusion proteins functionally...chloroplasts, the extracellular milieu, and gram negative bacteria periplasm. Efforts to tag endogenous ...

... mutated to a negatively charged residue, aspartate or glutamate, imparting the negative charge associated...smaller, and carry only a single negative charge as opposed to a double negative charge in the phosphate group...charges this tRNA with phosphoserine A specialized elongation factor that allows proper extension and translation...Sep-tRNA synthetase, and EF-Sep – an optimized elongation factor that accepts Sep-charged tRNAs during ...

... recombination has not occurred. This powerful negative selection system ensures high editing efficiency.../gRNAs are easily inserted using BbsI and Golden Gate Assembly. Either plasmid can be also linearized ...repress transcription by physically blocking the elongation complex from binding the DNA or extending the...activation of the system. Mobile CRISPRi for Gram-negative and Gram-positive bacteria Jason Peters, Oren ...different genetic systems, targeting either Gram-negative bacteria or Gram-positive firmicutes, to bring...introduced (Tn7 in Gram-positives and ICEbs1 in Gram-negatives) but share the common CRISPR-dCas9 system for ... Sharp JD, Xu R, Prisic S, Husson RN (2016) Investigating essential gene function inMycobacterium tuberculosisusing...

...electric current applied to the gel causes the negatively charged proteins to migrate toward the positive...binding.  Whenever possible, include positive and negative controls. Positive controls typically include ...relevant levels (not overexpression systems) while negative controls are those that do not naturally express... on the membrane the antibodies are typically conjugated to an enzyme, such as horseradish peroxidase ...protein of interest, is not labeled. Instead, a conjugated species-specific secondary antibody is used to..., take care to include the proper positive and negative controls, choose antibodies that are validated...

... of CRISPR technologies, one might incorrectly relegate RNAi to history. The straightforwardness of RNAi..., gene knockout strategies cannot be used to investigate essential genes. Historically, gene silencing...present at the first stage of an RNAi-based investigation. Creation of the RNAi trigger requires a few... which allows easy creation of long hpRNA with Gateway-based cloning and straightforward positive selection...assembly of intron-spliced hpRNAs through Golden Gate cloning. Artificial micro RNA and short hairpin ...from Worcester Polytechnic Institute where he investigated how plants achieve polarized cell growth in ...

...Overexpressed proteins also have the potential to form aggregates and may be toxic to the cell. Here comes the ...Initially, Tanenbaum et al. observed some GFP aggregation, which they reduced by using superfolder GFP ...expression levels of SunTag constructs would avoid negative effects on cell physiology. Having seen that mitochondrial...Since the v4 system does not display protein aggregation, it’s recommended for most imaging applications...with SunTag. v1 SunTag exhibits some scaffold aggregation (v4 Suntag does not.) It’s up to you to determine...