...insert (blue-white screening). Other types of screening methods Although blue-white screening is probably the... that! (To read about other screening strategies, check out our screening overview blog post.) *Note:...good, but how does this help us screen plasmids? Blue-white screening in the lab Scientists discovered... few common vectors. Blue-white screening is just that – a screening process. It does not select only...can talk about an even more specific method of screening your cloning reaction. Being able to select for...Note: Two Popular Vectors for Blue White Screening Available at Addgene Are: pUC18 and pUC19 Let’s begin ...from the lacZ gene (a mutation called lacZΔM15) creates a non-functional β-galactosidase enzyme. Providing...

...cell-type-specific Cre, the polyA sequence is inverted only in the Cre-expressing tissues. This inversion creates a template...studying the CNS and PNS! What is the CREATE method?  CREATE stands for Cre Recombinase-based AAV Targeted Evolution...using this library to infect an animal expressing Cre in a cell type of interest, a scientist can isolate...28671695. 2. Deverman BE, Pravdo PL, Simpson BP, et al. Cre-dependent selection yields AAV variants for widespread...rounds of infection and screening by PCR enrich for capsid variants with increased ability to infect the...to reach the brain. Delivering the CREATE AAV library via IV screens for capsids that are able to cross...that determines tropism) have been developed that increase transduction efficiency, none allow for simple...

...to provide them to their students. My desire to create a set of low-cost, easy-to-use educational biology...fragrances that they can smell, or enzymes that can create hydrogels that they can touch. With BioBits, we...

...in a sample. Digital PCR assays for mutation screening Now that we have introduced how digital PCR works...the bi-allelic mutations typically desired when screening clones for complete functional knockout of your...interested in harnessing the power of digital PCR screening to validate your genome edits, this paper provides...Remember that although digital PCR is a great screening tool, it still requires target DNA sequencing ...PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells. PLoS ONE 11, e0153901–...

...worked with high school students in the bay area to create a plasmid rap video for us (If you’re new to plasmids...unfortunate story of Rosalind Franklin and her incredibly important role in solving the structure of DNA...is a YouTuber and science educator looking for creative ways to implement the next generation science ...

...CRISPR knockout screens: Full genome vs. custom screens Full genome CRISPR knockout screens have been used... gRNAs in a screen, to ensure that each gRNA has a good chance to contribute to the screen. In the end...Secondary screens  If you have generated a list of candidate genes from a full genome screen, a smaller...genes you’d like to investigate in your next CRISPR screen. Luckily, the Green Listed software can help you...software tool used to design gRNAs for custom CRISPR screens targeting a (long or short) list of genes provided...fantastic tools for unbiased discovery. With these screens, the investigator generates a cell population where...involved in the studied phenotype. For robustness of a screen, each gene is not targeted by only one gRNA, instead...

...means to create barcoded gRNA libraries that can be used to combinatorially modify the genome, screen for ...revolutionized how we decode the genome by making it easy to create specific genetic perturbations. The ease with which... off-target activities have proven themselves incredibly useful for the efficient generation of individual...multiplexed CRISPR systems for high-throughput screening are vastly useful for mapping the combinatorial.... Hart, Traver, et al. "High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer...Hiroko, et al. "Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide...PMC4420636. 7. Ma, Hongming, et al. "A CRISPR-based screen identifies genes essential for West-Nile-virus-...

...advantage of these discoveries to create a new system for screening protein-protein interactions. In later...identifying prey proteins after screening. It is also possible to screen libraries of bait and prey against...single-pass two-hybrid screening (6). See the Tips section for more on how to increase your rate of interactions...interactions when screening! Tips for two-hybrid screening Minimizing false positives: Run replicates of ...cerevisiae in 1989 and are still used extensively to screen for molecular interactions in the cell, including...protein interact and enable intein splicing (5). Screening with yeast two hybrid systems While initial studies...use many preys with a single bait in Y2H-based screens. In this way, proteins that interact with a molecule...

...transgenic models that express recombinases like Cre or Flp in their cells of interest and recombinase-dependent... pairs of specific sequences, lox (recognized by Cre) or frt (recognized by Flp), to either flip or excise...The two recombination sites are both recognized by Cre, but they are comprised of different sequences (loxp...lox2272) and can not recombine with each other. When Cre is introduced to the system it binds one pair of ...sandwiched between 2 loxp sites and is excised by Cre (Figure 1). This results in one loxp site and one...of this spurious expression?   Figure 1.  Cre-recombinase binds to either pair of lox sites, let's...to leaky expression in cells that do not express Cre or Flp. Here at Addgene we quantify the amount of...

...DIO. Lox sites are recognized by the recombinase Cre. Cre first flips the sequence between the first pair...them.  Lox sites Lox sites, in combination with the Cre recombinase, are used to spatially and temporally...flipped transgene. By restricting the availability of Cre, lox sites lets researchers control the spatial and... activation of a transgene. Figure 2: FLEX Cre-Lox system.  WPRE The Woodchuck Hepatitis Virus...which aids in nuclear export of mRNA and helps increase the expression of your transgene. The WPRE is ...

... work more creative. An example could be making lab meetings more interactive and creating space for discussion...tools for creative interaction During Zoom meetings, different tools can be used to promote creative interaction...make the screen-sharing experience more interactive. The button is on the right of the Share Screen control...this is an underrated ritual that really drives creativity and novelty in scientific research and in any...Virtual lab meeting? No problem! You can still creatively collaborate and brainstorm! Image from Joseph...control panel on the top of the screen and every participant get to annotate things as they come up in their...their minds. The final screen can also be saved and exported as a picture. I believe that showing something...

...Cervical cancer screening in Ghana Gladys is currently working to enhance cervical cancer screening in Ghana....cervical cancer screening for a limited group of Ghanaian women by including PCR screening for HR HPV alongside...as positive controls in her PCR screens. She hopes that these screens will both identify individuals who...to be at high risk by these screens can then undergo more routine screening allowing for early cervical...positive controls in PCR-based cervical cancer screens. This third grant represents the continuation of...back in June, Gladys will be using plasmids in creative ways that we wouldn’t necessarily have expected...tests are commonly used in Ghana, there is little screening for DNA markers like the presence of high risk...

...payment for working more than 40 hours per week will increase from $23,660 to $47,476 per year, under updates...2014, the White House issued a memorandum to the Secretary of Labor to update the FLSA, which had not seen...automatically adjusted every 3 years. In May 2016, Secretary of Labor Thomas Perez updated the overtime regulations...post by Director of the NIH Francis Collins and Secretary of Labor Thomas Perez, Fair Pay for Postdocs: ...made by multiple organizations across academia). Increased postdoc wages, overtime tracking? What are institutions...should therefore rise from $43,692 to $47,484, an increase of 8.7%. However, it is hard to gauge exactly ...negotiated a raise from $43,692 to $48,216, an increase of 10.4%. The Icahn School of Medicine at Mount...

... vivo screens in naïve T cells. More broadly, this system can be used to perform in vivo screens in other...power of in vivo screens. Figure 1: Using FITS to perform in vivo CRISPR screens to assess T cell...FITS In vitro CRISPR screens in immune cells have identified many putative regulators of immunity. However...However, in vitro screens cannot faithfully model the complex spatial, cellular, metabolic, and cytokine...vivo. Over the past five years, in vivo CRISPR screens have been used to interrogate T cells, an important...complex and physiological microenvironments. These screens have primarily been performed in activated T cells...naïve T cells. The Framework for In vivo T cell Screens (FITS) approach (Milling and Markson et al., 2024...

... your selection, then screen Why add a screen to your cloning strategy? A screen will help you more easily...a look at a few screening methods. Blue-white screen A widely used method of screening is the blue-white...In many cases, you will need a screening strategy as well. In a screen, you aren’t killing off a portion...example on a plasmid, creates a functional 𝛽-galactosidase. Blue-white screens rely on vectors where ...blue-white screening blog post for more information.   Figure 1: Result of a blue-white screen. Image...game. By using a variety of screening and selection techniques, you can increase the chances of finding the...that contain your desired insert? This is where a  screen comes in. Let’s take a look at All_in_one_CRISPR...

...they need to use them in their own work, it also increases the visibility of both you and the original depositor...work later. We also hope that this feature will decrease redundancy. If you modify a plasmid from Addgene...out there Beyond these 3 big pushes, we try to increase your work’s visibility in a few other ways. Every...

... this technology.” GeCKO library for genomic screens Using lentivirus as the delivery vehicle, Neville...to enable both negative and positive selection screening. They and their colleagues including Feng Zhang...at a time,” Wang said. “It was very difficult to screen in a pooled fashion or to go after thousands of...expression. The Yusa lab has used their library to screen for genes involved in bacterial toxin resistance... is now possible to conduct systematic genetic screens in mammalian cells,” David Sabatini, a senior author...Library Reference page.    References: Genetic Screens in Human Cells Using the CRISPR/Cas9 System. Wang... (PubMed). Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem O, Sanjana NE, Hartenian...

...drive expression of Cre-mCherry and a second promoter to drive expression of a Cre-dependent fluorescent...will only result in cells where Cre-mCherry is also expressed. If Cre-mCherry expresses in photoreceptors...photoreceptors and horizontal cells, and Cre-dependent-GFP expresses in horizontal cells and ganglion cells, only...horizontal cells transduced by both AAVs would express Cre-mCherry and GFP.   Figure 2: Synthetic promoters...different design strategies: Strategy #1 (ProA): Create promoters with sequences upstream of the start ...cell-type specific genes from mice. Strategy #2 (ProB): Create promoters using phylogenetically conserved sequences...high cell-type specificity.  Strategy #3 (ProC): Create promoters by repeating mouse cell type specific...

...biological process. These screens provide an alternative to genome-wide RNAi screens, which although highly...similar pitfalls exist for CRISPR screens and how best to design these screens to controls for flaws. Recently...CRISPR knockout screen to analyze the following unanswered questions about CRISPR screen design. Unanswered...CRISPR/Cas9 screen design Are non-targeting guides an appropriate control? Many CRISPR screens use non-targeting...optimal conditions for CRISPR knockout screens, the Bassik lab created a human knockout library that has 10...library was validated via a growth screen and a ricin toxicity screen since essential and non-essential...The library performed well in both screens, with the growth screen having a 1% false discovery rate and...

...AAV-GfaABC1D-Cre-4x6T AAV5 Recombinases Stanley Thomas Carmichael New viral prep pAAV-EF1a-fDIO-Cre AAV5 ...Collection. These preps drive expression of either Cre recombinase or a fluorescent marker in the various...Recombinases Esteban Engel New serotype pAAV-hSyn-Cre-P2A-dTomato AAV PHP.eB Recombinases Rylan Larsen ...Larsen New serotype AiP13778 - pAAV-AiE0450h-minBG-iCre(R297T)-BGHpA (Alias: CN3778) AAV PHP.eB Recombinases...viral prep AiP13779 - pAAV-AiE0779m_3xC2-minBG-iCre(R297T)-BGHpA (Alias: CN3779) AAV PHP.eB Recombinases... New viral prep AiP13781 - pAAV-AiE0452h-minBG-iCre(R297T)-BGHpA (Alias: CN3781) AAV PHP.eB Recombinases...