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...References Espah Borujeni, Amin, Anirudh S. Channarasappa, and Howard M. Salis. "Translation rate is controlled...Scarless Modification of Proteobacterial Genomes." Applied and environmental microbiology (2019): AEM-00990...chromosomal gene replacement in Escherichia coli." Appl. Environ. Microbiol. 81.15 (2015): 5103-5114. PubMed...

... explain how to verify that your cells were appropriately edited for your desired mutation - insertion...frequency is 50%, then you could infer that approximately 25% of cells are likely -/-, 50% are -/+, and...sequencing confirmation. All knock-ins A method applicable to knock-ins both large and small is a software...

...polycistronic E. coli expression constructs. Some applications of plasmids from this kit include the production...can be engineered to bind specific RNA bases. By swapping out these different modular repeat segments, you...variety of functions via different fusions. Some applications of PUF protein fusions reported in the literature...

...pool has been sufficiently selected with the appropriate antibiotic so that every cell in the culture ...stable cells such that the next day they will be approximately 50–60% confluent. For 293T cells this is about...confluent or the medium is acidified (i.e., the medium appears orange/yellow if using a standard phenol red pH...counting the cells. When you ultimately transfer approximately 50–100 cells to the conditioned medium, it will...solution at a concentration of 5 cells/mL. Prepare approximately 10 mL of this cell solution for each 96-well...which you see growth. At this time, the cells will appear as colonies in the well ( Figure 1 ). You will ...

...tools Once you’ve selected the class of tool appropriate for your experiment, you’ll need to find a specific...Yizhar, O., & Deisseroth, K. (2012). Principles for applying optogenetic tools derived from direct comparative...

...popular choice for a variety of synaptic imaging applications. Find iGluSnFR4 plasmids here! Aggarwal, A....that they could reprogram the genomic target by swapping the homology arms for new targets. And, importantly... is scarless, unlike previous integrase-based approaches. Find STITCHR plasmids here! Fell, C. W., Villiger...

...to specific DNA sequences. Non-editing CRISPR applications direct catalytically dead dCas9 fused to a variety... publication at Addgene. Klann TS, Black JB, Chellappan M, Safi A, Song L, Hilton IB, Crawford GE, Reddy...Ruan K, Wang F, Xu G, Hu R (2016) A CRISPR-based approach for targeted DNA demethylation. Cell Discovery...

...for sphingomonads and other Alphaproteobacteria. Appl Environ Microbiol. 2013 Nov;79(21):6795-802. doi...cumate-inducible gene expression system for Bacillus. Appl Microbiol Biotechnol. 2019 Jan;103(1):303-313. doi...expression of genes for metabolic engineering applications in Saccharomyces cerevisiae.” FEMS Yeast Res...

...contains the expected size insert. By selecting the appropriate enzyme(s), one can either linearize a plasmid...interest (see below for recommended amounts) Appropriate restriction enzyme (see manufacturer's instructions...instructions for proper amount) Approrpriate restriction digest buffer (see manufacturer's instructions) Gel...others. When choosing restriction enzymes for this approach, it is often a good idea to choose two different...products on a gel and those clones with the appropriately sized products should have the correct orientation...

...container Personal protective equipment (PPE) appropriate for the biosafety level of the lab space Reagents... like the one in the picture above. Wear PPE appropriate for the lab space in which you are working. Even...that the centrifuge is clean and that everything appears to be working smoothly. Place your sample tubes... the centrifuge so that the pellets form in approximately the same place in each sample. Place the centrifuge...according to speed and time (and temperature, if applicable) listed in your protocol. Pro-Tip Spin speed ...

...et al (2). In this post, I’ll cover two great applications of the NLuc technology. The first couples NLuc...the coupled luciferase. This strategy was first applied using RLuc8, an engineered Renilla luciferase, ...fluorescence. The paper reports a wide variety of applications of the Enhanced Nano-Lanterns including monitoring...

...cross-team guy” at Addgene, an unofficial title that I happily embrace (and admittedly, I may have started that...identify as one.  But some time around 10th grade, I snapped out of the phase of my life that had me listening...you’re part of a team - it will just make you a happier person. What does a good team need? Besides that...

...have been increasingly popular in fundamental and applied research since their first use in the early 90’... at transferring the gene of interest to the appropriate cells, it was obvious that its safety was a disadvantage...either tTA or rtTA (FUW-M2rtTA). In this kind of approach, a population of cells might express only the ...

...than longer ones. Thus, you can determine the approximate length of a DNA fragment by running it on an ... bromide (EtBr) to a final concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock... bands will be differentially intense. If this happens, you can just soak the gel in EtBr solution and...Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel. A typical run...are usually referred to as ‘bands’ due to their appearance on the gel. Pro-Tip If you will be purifying ...

...huge innovation, there was a limitation to this approach. CBEs and ABEs are base transition editors. They...GBEs), Xueli Zhang’s lab, returned with a unique approach to improving C → G editing. Instead of fusing ...abasic site. In this post, it refers to the same approach as a CGBE. BER Base Excision Repair A cellular...

...chance that all the edits you want to make will happen.  There are many reasons to multiplex gRNAs, some...adjacent to the sites. When digested with the appropriate Type IIS enzyme, the unique flanking overhang...subsequent “bricks” to be added one by one. This approach is especially useful for combinatorial analyses...

...process is favored over the other. Cell conversion happens via: ReprogrammingA differentiated somatic cell...cells into another cell type. However, these two approaches differ in several ways. Below is a table listing...: 26042384. 27. Nejadnik, H., et al., Improved approach for chondrogenic differentiation of human induced...

...microcentrifuge. Remove the gel from the plastic wrapper and rinse with deionized water. Gently remove the...5 µL of the prestained protein ladder to the appropriate well. Pro-Tip When possible, skip one lane before...µL of each recombinant antibody sample to the appropriate well. Pro-Tip Leaving clear lanes between samples... Take a brightfield image of the gel with an appropriate imaging system. Figure 1 Recombinant antibody...Gels . Select Plot Lanes . One graph per lane will appear with peaks representing each protein band. Use ...

...the basics of how to weigh reagents. Wear the appropriate personal protective equipment (PPE) prior to ...contain the reagent while on the balance, and an appropriately sized container to put the reagent in. For some...container. Dispose of the excess material into an appropriate waste container if the material is biohazardous...biohazard solid waste container. Conclusion Appropriately weighing reagents is important to ensure the...

...Thinking about using antibody-based applications in your experiment...but not sure where to start or ...or quick refresher on — these common antibody applications.  Thanks for watching, and we hope you found...