...can find the subsequent transformation protocols below: OpenWetWare Transforming chemically competent ...Untergasser Lab Transformation of chemically competent cells (protocol)  Transformation of electrocompetent... easily transformed without a heat-shock step. Because of the large number of transformations being performed...that transforms well in other competent cells) to ensure that your cells can be readily transformed. Compare...Want even more tips for transforming your cells? Check out our transformation guide.   ...We perform over 100 transformations each week at Addgene, so we need to be cost and time efficient. We...a little-known reagent for streamlining the transformation step. Read on to find protocols and tips that...

...reagents can be delivered via transfection, nucleofection, viral transduction or injection as either protein...cell line that can be transfected at high efficiency. Lentiviral transduction Cas9 and gRNA can be present... present in a single lentiviral transfer vector or separate transfer vectors. May contain reporter gene...inserted into an AAV transfer vector and used to generate AAV particles. Transient or stable expression...Chemical transfection   Lipid mediated Cationic polymers Calcium phosphate Cells Transformed cell lines... lines Transformed cell lines Transformed cell lines Cargo Nucleic acids or protein Nucleic acids Nucleic... method: Do you need stable integration or is transient expression sufficient? Which cell types will you...

... not appear to mind extraneous proteins bound to its outer mitochondrial membrane. Sequestration is accomplished...Updated mini-transposon vector for bacterial mutagenesis or gene targeting Victor de Lorenzo's lab...modular mini-Tn5 vector that can be used to generate random mutagenesis libraries or to insert heterologous...selecting the important elements from existing transposon and vector systems and creating an all-synthetic...validated this vector, called pBAM1, by conducting random mutagenesis in the soil bacterium Pseudomonas putida...family of GFP proteins in the cephalochordate Branchiostoma floridae, named bfloGFPs. They went on to characterize...mitochondrial turnover Mitochondrial health, transport and turnover are important features to be monitored...

... could infect. Finally, the transfer plasmid encoded the desired transgene flanked by HIV-1 long terminal...integration. After co-transfection of these plasmids, 293T cells released transgene-containing lentiviral...protein), Pol (polymerase), Tat (viral transactivator for transcriptional activation from the 5’ LTR) and Rev...capacity of a lentiviral vector but for which transient transgene expression is sufficient. Lentiviral vector...include a large genetic capacity and the ability to transduce both dividing and non-dividing cells. Lentiviral...integrate into the genome, permitting long-term transgene expression, but they could only infect actively...infect non-dividing cells, but without sustained transgene expression. To design a viral vector system that...

...PHP.B has a >40-fold increase in CNS transduction and transduces the majority of astrocytes and neurons... mice whose CNS was transduced by PHP.B but absent in mice who were not transduced by PHP.B. The Deverman...These results were surprising because in vivo transduction of AAV serotypes generally tracks between strains...diverse library of AAV9 mutants that had a small randomized peptide loop inserted in the AAV9 capsid. This... an enhanced version of PHP.B, shows similar transduction efficiency, but at a log lower dose than PHP.B...permissive and six as non-permissive to PHP.B CNS transduction. Two genes were associated with the PHP.B permissive...outbred mice (F1) had half the level of CNS PHP.B transduction as their C57BL/6J parent. The second generation...

...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: ...

...Reverse transcription of the transgene occurs in the cytoplasm, then the resulting double-stranded DNA transgene.... However, IDLV genomes are transcriptionally active, leading to transient expression of gene products...lentiviral vectors is random, and the integration site will be different for each transduced cell, there’s no...mutagenesis (Howe et al., 2008). 2. Transient expression of transgenes/shRNAs If you want your cells to ...They rely on a viral protein called reverse transcriptase to convert their RNA genomes into DNA to be ...second-generation system as an example.  The lentiviral transfer plasmid encodes your insert of interest, flanked... larger protein: PR (protease), RT (reverse transcriptase), and IN (integrase). These viral packaging ...

... 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...