...libraries, chemical transformations can also be used. Efficiency for chemical transformations is less than that...for your transformation(s). The amount of competent cells you need total and per transformation may vary...the manufacturer’s protocol for transformation and expected transformation efficiency, or more specifically...with a large backbone might be more difficult to transform.    Figure 1: Factors to consider when ... coverage, since this will help decide what transformation efficiency you need to achieve. Most pooled...contains 5,000 plasmids, your goal for your transformation would be to obtain 100 x 5,000 = 500,000 colonies...likely rely on serial dilutions to estimate transformation efficiency (Figure 2).    Figure 2: ...

... the transgene of interest. In our studies, mCherry interfered with the transport to the membrane of the...the hM4Di might improve the transport of this DREADD to the plasma membrane. The haemagglutinin (HA) tag...the expression of the transgene of interest and its localization in the membrane. The lesson we learned...2013) could help improve the transport of protein of interest the membrane.   Figure 1: Electron... the plasma membrane. Note that the use of the HA tag significantly improves the transport of hM4Di to...versatile method to identify the expression of transgenes of interest. Furthermore, fluorophore variants...only a small proportion were found on the plasma membrane (top panel in Fig. 1). Of course, this was problematic...

...tetracycline operators. Transcription can be turned on or off depending on what tet transactivator is used. UAS...for in vitro transcription, the promoter drives either the sense OR antisense transcript depending on ...This element is responsible for initiating the transcription of your insert into RNA. In practice, the term...controls the binding of the RNA polymerase and transcription factors, therefore promoters play a large role...expressed.      The RNA polymerase(s) RNA is transcribed from DNA using an RNA polymerase (RNAP). In bacteria...

... differences between transformation, transduction, conjugation, and transfection Resources on Addgene.org... in 3 different ways: Transformation grade DNA Cloning grade DNA Transfection grade DNA Figure...Figure 1: Transformation introduces transformation grade plasmids into a bacterial cell. Transformation...bacterial transformation of chemically and electrocompetent cells is minimal (picogram range). It can ...plasmids into mammalian cells? Use transfection grade DNA. Transfection is the process of inserting an ...Transformation grade DNA  Transformation is the process of inserting exogenous DNA in a microorganism. The amount ...without commercially available kits and DNA for transformation doesn’t need to be as pure as for other uses...

...project forward, transductions should not be rushed into. Before you start any transduction, you should always...Primers targeting the transgene are also beneficial as they ensure that the correct transfer plasmid was used... positive for vector encoded transgene expression following transduction with serial dilutions of a viral...resistance genes carried on transfer plasmids. Target cells are transduced with serial dilutions of a ... your packaging cell line, prepped your DNA, transfected and harvested your lentivirus. Now it’s time ...assaying for expression of a gene carried on the transfer plasmid or quantifying the number of viral copies...gag, WPRE, antibiotic resistance-genes, or the transgene itself. Many researchers prefer to design primers...

...they cannot cross cell membranes, so they are best used either with plasma membrane proteins (such as GPCRs... minimum energy transfer values you can expect, thereby establishing the dynamic range of your FRET assay...what's it good for? Fluorescence resonance energy transfer (FRET) is a biophysical interaction between two...first fluorophore (the donor) is excited, it can transfer energy directly via resonance to the second fluorophore...structural changes in the ribosome during protein translation (Ermolenko et al., 2007). All that is required...develop a successful FRET-based assay since energy transfer only occurs when donor and acceptor are close ...Å away from the donor doesn’t result in energy transfer and thus, your specific FRET signal is unaffected...

... polymerase and transcription factors. Since the promoter region drives transcription of a target gene...promoter to induce transcription of the phage repressor cI. Repressor cI inhibits transcription from phage promoter...lag time due to the time needed for transcription/translation. Many thanks to Nicole Zurcher who contributed... the promoter, turning it ON and initiating transcription.     Negative inducible In the OFF state, the...a bound repressor protein actively prevents transcription. Once an inducer binds the repressor protein...the DNA. With the repressor protein absent, transcription is turned ON. Types of inducible promoters Chemical...activator rtTA (reverse tetracycline-controlled transactivator) is normally inactive and cannot bind the tetracycline...

...packages have spent days to weeks in transit and some have been transported across oceans and continents. Each... treated to easily take in plasmids (transformation). Transformed bacteria are then grown up on antibiotics...received is immediately transformed and stored. Some samples need to be transformed with a micropulser using... performs an average of 140 transformations. The lab typically transforms newly deposited samples from...delivered by a mail courier and then the journey of transformation and storage begins. Some samples are submitted...long-term storage in our facilities, every sample is transferred to a 2D barcoded tube (see next section) and ...make a new tube from a replicate sample. The transformation After the DNA has been received and stored,...

...-visual G protein signal transducers (collectively known as the transducerome) that can be activated downstream...Resonance Energy Transfer (BRET). BRET is similar to Förster Resonance Energy Transfer (FRET), in which...non-visual G protein transducers. To use the TRUPATH system, cells are transfected with plasmids expressing...including smell, taste, sight, inflammation, and neurotransmission. But with approximately 950 different human...involved in sight (Olsen RHJ et al., 2020). A transducer complex consists of a trimer of three subunits...such a tool is being able to identify specific transducer complexes that are activated in response to a...fluorophore. The difference for BRET is that the energy transfer is between a luminescent donor and a fluorescent...

...Selectable transgene markers for plant transformation fall into two main categories: antibiotic/herbicide...presence of a transgene instead, which can’t be done if your selection method kills the transgene free individual...Arabidopsis seed following floral dip transformation next to an untransformed seed from the same plant beginning...resistance genes usually involves killing the untransformed seedlings or callus (undifferentiated plant ...For example, with CRISPR-Cas9 gene editing, transformation may be identified by resistance to an antibiotic...antibiotic. Successful editing of transformants then typically occurs in T0 plants (from callus) or T1 plants...contaminate it. I have used it for selecting transformation events and am currently planning to use it ...

...their position relative to a transcription start site. In STARR-seq, randomly sheared genomic libraries ...of these random stretches of DNA contain an enhancer, it will activate its own transcription from the ...alone. Molecular processes from signal transduction to transport rely on protein-protein interactions and...an alternative method, called STARR-seq (self-transcribing active regulatory region sequencing) that tests...single gene. The gRNA expressing cells were then transfected with a Cas9-expressing plasmid to generate a ... Receptor-ome Expression and Screening via Transcriptional Output-TANGO (PRESTO-TANGO) to screen the NCC...RAS signaling pathway and includes 180 unique transcripts. Each gene was chosen to represent the primary...

...light-dependent activity to transcription, they fused BldD to the transcriptional activators p65 and VP64 ...readily found at Addgene. Shining light on transcriptional activation using dCas9 Initial photoactivatable...early 2015 focused on using light to control transcription. Two separate labs, Moritoshi Sato’s lab at ...photolyase homology region (CRY2PHR) fused to a transcriptional activator domain (VP64 or p65). Upon expression...the activator into position to activate gene transcription. The researchers tested a variety of combinations...CRY2FL-VP64, a fusion of full-length CRY2 and the transcriptional activator domain VP64. Using this system in...(Nihongaki, et al., 2015) resulting in a double strand break (DSB) that can be repaired by either non-...

...specific titer, you can better plan your transductions and use a range of reasonable MOIs. 2. Create a monoclonal...generated from A549 cells transduced with lentiCas9-blast. A549 cells were transduced (MOI = 37) and selected...generate DNA duplexes with one strand from the edited cells and one strand from the control cells, which.... The appearance of the 428 bp band indicates edits in the BRAF gene in the BRAF-gRNA transduced cells...generated A549 cells stably expressing Cas9 via transduction with Cas9 lentiviral vector, lentiCas9-Blast... Figure 1: Cas9 expression in A549 cells transduced with lentiCas9-blast. Anti-Cas9 Western blotting...whole-cell extracts from stable pools of A549 cells transduced with Cas9 expressing lentivirus at the indicated...

...can be motifs within transmembrane proteins, such as receptors, or post-translational modifications on those...diffuse through the cell membrane freely, while others require a permeabilized membrane to enter cells. Depending...label intranuclear proteins of interest, you can label cytosolic proteins alongside the intranuclear ones...Depending on the application, they can be membrane-permeant or membrane-impermeant, and the cells of interest...viability dyes are membrane-impermeant and can only enter cells with compromised plasma membranes — i.e., dead...conjugates are commercially available for a wide range of fluorophores, allowing for great flexibility ...epitope-targeting antibody can originate from a range of species; for example, rabbit or mouse. In a second...

...DNA must first be transcribed into messenger RNA (mRNA). mRNA can then be translated at the ribosome into...consists of transfecting cells with your DNA template of choice and allowing these cells to transcribe, translate...expression post-transfection. Mammalian systems can express proteins both transiently or through stable...expression vector that is then transformed into a bacterial cell. Transformed cells propagate, are induced...vitro using purified components of the transcription and translation machinery. These include ribosomes, ...proteins are then modified via an array of post-translational modifications including protein folding, formation...proteins from mRNA and the addition of post-translational modifications Learn more molecular biology ...

...in E. coli BL21(DE3) as transformants failed to grow after the transformation of pTargetF into the BL21...pTnsABC expresses the transposases TnsA, TnsB, and TnsC pQCascade expresses the transposase TniQ, the Cas proteins...expression transposases leading to increased integration frequency. (C) Increasing rounds of transfer on transposition-inducing... engineering single genome edits but the double strand breaks it introduces makes it difficult to perform...chromosomal integration using CRISPR-associated transposases” or MUCICAT. Here, we’ll describe each of these... This would result in the lack of recovered transformants. Our speculation motivated us to update the ... sequence in pEcgRNA. Next both plasmids are transformed in E. coli with the donor DNA for editing to ...

...strategy uses a transposon called Tol2 to randomly integrate foreign DNA into the genome. Non-random integration...et al., 2019). Generating zebrafish transgenics Creating transgenic organisms by introducing foreign DNA...Danio rerio) have been used since the 1930’s in a range of biological studies, including investigations ... to produce abundant, externally fertilized, transparent progeny (Figure 2). These features give it a ...individual to maintain. Also in contrast to mice, the transparent zebrafish embryo develops outside of the mother...sequenced genome also permits “whole-genome/transcriptome” studies, in which, for example, the activity...mutants In the 1990’s, two revolutionary large-scale random mutagenesis screens produced ~1,500 zebrafish mutants...

... a strong transcriptional activation domain. Guided by dCas9, VP64 recruits transcriptional machinery ...as two other potent transcriptional activators (p65 and Rta). These transcriptional activators work in ...is fused to CBP, an histone acetyltransferase domain capable of rearranging chromatin structure. This method...interest. Upon binding, CRISPRa systems recruit transcription factors to increase gene expression. CRISPRa...CRISPRa methods vary in their transcriptional activators: some methods rely on fusion proteins while others...gene regulation. This can be used to activate transcription during either initiation or elongation, depending...uses specially engineered sgRNAs to increase transcription. This is done through creating a dCas9/VP64 ...

...previously require a preliminary double stranding step. The double stranding step was assumed to be necessary...genome is single-stranded and both TA-based ligation and tagmentation require a double-stranded template.  ...extraction, the [+] and [-] strands might naturally associate forming a double-stranded species that could be...extract is a heterogeneous mix of single-stranded and double-stranded species. While not all of the molecules...extract associate with complementary strands, enough double-stranded species exist in the mix to serve as...viral genome We hypothesized that this double stranding step might not actually be necessary. Since the...the AAV genome exists as [+] and [-] strands that are packaged at the same frequency, we reasoned that...

...), YFP (yellow), and membrane-tethered CFP (cyan). Brainbow 1.1 adds OFP (orange) (Livet et al., 2007)... mouse generates cells that randomly express either nuclear GFP, membrane CFP, cytoplasmic YFP, or cytoplasmic...labeling technique involves the introduction of a transgene construct containing fluorescent proteins (XFP...Because each cell can have multiple copies of the transgene that will recombine independently, cells may acquire...respectively, cells will fluoresce either red or orange until recombination occurs. Following the development...incorporates the highly photostable fluorophores mOrange2, EGFP, and mKate2 (red) to solve the problematically...non-fluorescent form of YFP as the default state of the transgene such that recombination is necessary to excise...