... the library are differently sized, smaller plasmids may be amplified preferentially, skewing the composition...some of Addgene’s most exciting and versatile offerings! We recently re-amplified our distribution stock...successful amplification? To provide a point of reference, Addgene commonly prepares NGS samples using both...overrepresented plasmids (plasmids that were amplified preferentially). You can also rank the plasmids by abundance...

...illustrates the utility these tools have in many different areas of biology as they grow. The top 15 plasmids...envelope protein enables this virus to infect many different cell types so it is useful in a wide range of ...lentiCRISPR v2 - This 3rd generation lentiviral transfer vector also from the Zhang lab is used to deliver... David Root lab is a 3rd generation lentiviral transfer plasmid for cloning and expressing new shRNA sequences... Jennifer Tsang...

...organizers. My overall impression from this conference was that Cancer Researchers and Immunologists...Here I aim to compile a few highlights from the conference which reflect how cancer/immunology researchers...multi-pronged attack. Inflammation and tumorigenesis The conference opened with a very interesting presentation by...our Fluorescent Protein Guide. Throughout the conference aspirin and the use of COX inhibitors was discussed...about our free deposit and distribution service! References: 1. Pietrocola, Federico, et al. "Caloric restriction...

...mutation of your favourite gene, there are several different methods available. Thankfully, there are many ...mutations. The assays you will use aren’t that much different from conventional duplexed primer probe assays...primer to amplify the target locus, as well as a reference probe designed to bind at an unedited site distal...are completely “blind” to this very important difference (Figure 3). If you are interested in harnessing...biology. Follow him on Twitter @AnotherLabRat.     References 1. Miyaoka, Y. et al. Isolation of single-base...

...AAV8) enable different patterns of transduction and transgene expression across different cell types (...world. We began with only a few inventory items offered domestically, but by the end of 2016, we expanded...visualizing hM4D(Gi)-mCherry in fixed tissue. References 1. Aschauer DF, Kreuz S, Rumpel S. Analysis of...Kilic E, Bähr M. Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression...

...You’ll want to pick enzymes that will give you different sized bands depending on whether the plasmid contains...using vector-specific primers, look for size differences. You can also use primers where one primer anneals...popular sequencing primers in our Molecular Biology Reference. Sometimes common  primers can't provide all of...Sanger sequencing Read the Molecular Biology Reference ... Jennifer Tsang...

...These cells are then transfected with several different constructs for the reporter to work and to make... visual read out allows for easy screening of different gRNAs by simply changing the gRNA used when transfecting...concept the Denham lab compared editing at three different genomic sites in the AAVS1 locus using GEmCherry2...GEmCherry2. In this experiment they saw a clear difference in mCherry expression using FACs sorting, confirming...mutant of interest, give these methods a try.  References Højland Knudsen C, Ásgrímsdóttir ES, Rahimi...

...commonly used in research laboratories to detect the different subclasses, but there are also secondary antibodies...secondary antibodies to detect multiple targets with different mAbs in the same experiment. The Trimmer Lab found...within the plasmid backbone to generate R-mAbs of different subclasses, including subclasses that were not... useful research materials and information. References and Resources Additional Resources on the Addgene...Addgene's recombinant antibody plasmid collection References Andrews NP, Boeckman JX, Manning CF, Nguyen JT...

...the answer isn't to work harder - it's to work differently.  Tools to Try  Checklists Found in surgical ...and your workflow can have a huge impact. Test different approaches, like whether it's better for you to... more than anything else I tried, made a huge difference in my ability to focus, especially on days when...what can be a rather vicious cycle.  Everybody’s Different   The truth is, there is no One True Way to Error-Free...the detail-driven daily grind of science.      References and Resources Images used Roberson Lab, UPenn...

...Pro tip! If your samples and control underwent different treatment processes, you may see a shift in the...presence or absence, the plots can look quite different. You might have high expression, low expression... the mean fluorescence intensity (MFI) of the different populations. MFI is the average brightness from...plot. However, the histogram plots look a bit different than they do for yes-no readouts.      ...above is for a smeary expression and is quite different from the yes-no histogram plot shown previously...

...DNA. The other component of these systems, methyltransferases, methylate DNA at particular sequences to...restriction modification systems containing methyltransferases that add methyl groups to specific DNA sequences...contain 3 methylases that recognize and methylate different stretches of DNA: Dam methylase adds a methyl...plasmids with no methylation (there are no methyltransferases in the PCR reaction). The template plasmid...could overlap with a methylation site. The quick-reference table below lists 10 common enzymes that may be...

...to give you advice and guidance. Second, I will offer some advice on how to “make the ask” once you have...have a “posse” of advisors who can help us in different ways and in diverse areas. It is helpful to have...information in these searchable databases are usually offering their contact information so you can tap them ...someone you trust. A good coach can make a big difference, if you can find one. Create your own peer mentoring...with progress. Tell them how their advice made a difference, helped you make a change for the good or resulted...

... will be depositing multiple plasmids and would prefer to send us your plasmid information in spreadsheet...plasmids all built with the same backbone and only differing in the gene/insert. Pooled libraries are normally...click the "Save" button and jump around to the different pages by clicking on the appropriate boxes at ...at the bottom of the page before moving onto a different page so that any data you enter is not lost.   ...to the backbone, and the vector type. Pro Tip! Reference the Addgene ID in the "Backbone Manufacturer" ...concentration of 0.1–1 µg/µL (or bacterial streaks, if you prefer). If you wish to edit your plasmid data after submission...

...mammalian systems has been in question. Three different labs have recently published work that provides... gene activation  and base editing, whereas a different set of mutations (the “V3.1” version) works best...miniature Cas system that utilizes a Cas12f from a different species, Acidibacillus sulfuroxidans, AsCas12f1...They further develop a version of SEND for gene transfer with pseudotyped viral particles, expanding options... the Liu Lab found that mismatch repair (MMR) interfered with the desired resolution of prime editing ...

...protein expression journey! Resources and References References Nabet et al. The dTAG system for immediate...turn off expression for good, the Cre-Lox system offers inducible yet permanent gene excision. Essentially...days. There’s also a CRISPR for that! CRISPR interference (CRISPRi) targets a catalytically dead Cas9 ..., and expression is tunable by titration with different amounts of tetracycline. Tetracycline regulation...

... put your experiments Nsync.   References and resources   References Dolan, E., Wang, Hou, Zhonggang,...while also highlighting their similarities and differences to Cas9. DNA targeting Cas enzymes Cas9 – The...Originally classified as Cpf1, Cas12’s primary difference from Cas9 is that it generates staggered breaks...(RNA knockdown, RNA editing, etc.). The main differences between Cas7-11 and Cas13 are 1). non-specific...

...billions—but when drugs fail, they cause human suffering and death. Doing research right has the potential... relevant? Research shows that there are sex differences in the therapeutic capacity of stem cells. Yet...male stem cells. Nor did they detect important differences in function between male and female cells. The...in basic research.  In addition to the studies referenced above, another excellent resource for research...population. To learn a little more about where differences in effects on the sexes come from, one clue or...

...plasmid) and any other important features that differentiate this plasmid from its predecessor. Important...production and storage, we grow a little extra and transfer 100 µl of glycerol stock to each of 2 96-well ...sequencing provider. Each sequencing service will have different requirements for volume and concentration, and...starts by determining what features you expect to differ between plasmids, and selecting or designing a ...Resources on the Addgene Website View our Plasmid Reference Pages Get Plasmid Backbone Information from Our...

...GBPb-GAL4 (DNA-binding domain); and a UAS-driven luciferase reporter construct. In the absence of GFP, no...create a complete transcription factor, activating luciferase transcription. Tang et al. coined this system... such that they activate gene expression from different promoters. Moving to an in vivo system, Tang et...eager to see how you use them in your research!  References 1.Tang, Jonathan C.Y., et al. “A nanobody-based...plasmids from this publication at Addgene. 3. Kirchhofer, A., et al. “Modulation of protein properties...

... 1-4 different gRNAs. Viral envelope proteins To alter the cell tropism of Nanoblades, different viral...embryos, and animals. Nanoblades can also carry different types of CRISPR cargo including RNPs for indel...can then fuse with the target cell membrane, and transfer their CRISPR cargo into the zygote. As a proof-of-principle...versatile and accessible genome editing tool.   References Mangeot, Philippe E., et al. "Genome editing ...