...reasons, it is essential to test the selectivity of your antibody. But testing is not one size fits all....important to test for the protocol you intend to use in your experiment. An antibody that demonstrates selectivity...best to determine this by testing them. For intracellular targets, we usually test 3 different fix/perms ... Correctly interpreting your data therefore necessitates careful design of RNAi sequences, confirmation...like a blood sample containing many different leukocytes. Where data is available from antibody-independent...quite useful. HCDM, among other things, works to test flow cytometry antibodies through their HLDA workshops... labeling patterns on different primary blood leukocytes. The file shared also contains information about...

...important for candidates during a job search and it is even more critical for international candidates. Being...international candidates that they should join their relevant associations/groups. These candidates should also...looking to continue their careers in the United States. There has been a growing shift away from employers...with filing a permanent residence case with United States Citizenship and Immigration Services (USCIS). If...years to work (up to 40 years for recent Ph.D. graduates). Therefore, it is imperative to plan for and ...following paths to maintaining a status in the United States including green cards that are well-tailored to...startups and firms with a non-international focus. Candidates in need of an industry H-1B need to be aware ...

...following a report that one can use linear repair templates (ie. PCR-derived dsDNA) with 30-60 basepair homology...ribonucleoprotein complex with linear DNA as repair templates which reduces time to generate edits to only four...rescued F1 animals (gel image under worm). Image courtesy of Jordan Ward, UCSF. In 2015 (Ward, 2015)...optimize a range of editing parameters: oligo repair templates with homology arms of 35-80 bp, and DNA double-strand...guidelines for designing single-stranded repair templates (Farboud et al., 2019).   The Mello lab also reported...of oligos used to generate linear dsDNA repair templates by PCR boosted efficiency (Ghanta et al., 2018...that melting and rapidly cooling dsDNA repair templates dramatically boosted editing efficiency (Ghanta...

...profile) and can also be used as a paternity test. The test is accurate, precise, robust, and STR profiles...for a range of testing, from animal doping to forensics, DNA sequencing to drug testing. You can find ...favour of biochemical assays followed by in vivo testing. The mantra being “you can’t trust those whacky...their origins. This technique is still used today to test for HeLa contamination. HeLa contamination is still...closely followed by T24 bladder cancer cells. Estimates of the amount of money wasted in basic oncological...Down-Regulation of HIF-1α by NDRG2 Expression Attenuates Proliferation and Invasion in Renal Cancer Cells...directly isolated from patients. In these cases, a test known as Short Tandem Repeat (STR) profiling can...

...were designed to target genomic sites with no annotated function, i.e. sites that lack open chromatin marks...20,500 protein-coding genes. Unique, non-overlapping sites in the genome were targeted and predicted on-target...binding site, or a coding region. Targeting these sites should control for the effects of guide expression... 3) safe-targeting guides, which target genomic sites with no annotated function. In the growth screen... safe-targeting guides were depleted at greater rates than their non-targeting counterparts, suggesting...screen Next, the toxicity of guides with off-target sites was profiled. Guides with exact or 1-bp mismatch...off-targets were only toxic if they had 5+ off-target sites. Results from the screen also reproduced several...

... the backbone vector has multiple BbvCI sites. If these sites have opposite orientations, then both strands...the sites are in opposite directions, you just need to use a plasmid with BbvCI restriction sites in the...Scientists use deep mutational scanning to simultaneously test how multiple amino acid changes affect a protein...Hundreds of thousands of protein variants can be tested in one experiment. There are many functional assays...choosing and validating a selection system. You can test the effect of multiple mutations in combination ...shuffling to combine pairs of mutations together to test combinatory effects. One-pot saturation mutagenesis...comprehensive mutagenesis library that’s ready to be tested in a functional screen of your liking. This method...

...2023. DNA damage drives genome instability and contributes to cancer, premature aging, and other harmful...displacement loop (D loop). The recombination intermediates can then resolve to complete the DNA repair ...a one-ended DSB). This single invasive strand initiates primary synthesis from a template followed by ...sequence to be modified. In general, the insertion sites of the modification should be very close to the ...determinant when selecting a type of donor. ssDNA templates (referred to as ssODNs (oligodeoxynucleotides)...insertions such as fluorescent proteins or selection cassettes. This class of template should have homology arms...1000 bps. HDR efficiency with circular plasmid templates is generally low; to increase the frequency of...

...fragments. When designed correctly, the recognition sites do not appear in the final construct, allowing for... the gene of interest is designed with Type IIS sites (such as BsaI or BbsI), that are located on the ...outside of the cleavage site. As a result, these sites are eliminated by digestion/ligation and do not ...final construct. The destination vector contains sites with complementary overhangs that direct assembly...transient construct retains functional Type IIS sites and will be re-digested. In contrast, formation ...construct does not retain the enzyme recognition sites. As a result, the ligation process is close to 100%...of fragments, and the loss of enzyme recognition sites after ligation favors formation of the construct...

... junction proteins cx43 GJB1 GJB2 Bioelectric states can alter cell types and patterning There is mounting... or cancerous it is (Levin et al., 2017). These states can even be necessary and sufficient for certain... the entire worm during regeneration, the worm creates two heads. Subsequent cutting results in a two-...visualize or perturb these slower-changing bioelectric states in living cells. Since bioelectrical signals are...indicators (GEVI) are ideal tools to track bioelectric states (Matthew et al., 2018). These allow for not only...only recording but also modulating bioelectric states in vivo. For example, the Levin lab from Tufts University...fusion of the voltage sensing domain of a Ciona intestinalis voltage sensor and a mutated version of the ...

...steadily increasing. Of course, you want the ‘latest and greatest’ variants with the best properties for your... affinity and a tendency to form higher order aggregates. This property can be tolerated in some applications...these experiments are discussed here. In vitro tests for aggregation The tendency of purified fluorescent...dimerization tendency. Another technique that separates molecules based on size is ultracentrifugation...of homodimerization in aqueous solution. In cyto tests for aggregation To what extent does homodimerization...cellular assays have been proposed. Early efforts tested whether or not FP-tubulin fusion proteins could...form microtubule networks in cells. Other efforts tested whether FP fusions to connexin localized correctly...

...transcript longevity in eukaryotes and promote transcript degradation in prokaryotes.  Prokaryotic termination...polyadenylation Unlike prokaryotes that have a single RNA polymerase for transcription, eukaryotes have three RNA...plays an important role in RNA processing and contributes to variability in RNA half-life, and ultimately...thought of as a eukaryotic-specific process, prokaryotes also add poly(A) tails to certain RNAs. Unlike...been noted that each eukaryotic RNA polymerase terminates differently. Polymerase III, for example, relies... to the Rho-independent termination found in prokaryotes. This is different than Polymerases I and II,...rbGlob) include the sequence motif AAUAAA which promotes both polyadenylation and termination. Out of those...

... lab built synthetic promoters from scratch and tested to see if they drove cell type specific expression...that are upstream of the transcription initiation sites of at least two genes with high expression and high...cell type specific transcription factor binding sites interspaced with random sequences.   Strategy #4...AAVs were first injected into the eyes of mice to test for cell type-specific expression of GFP. Half (...promoters that are also unlikely to express in primates and humans. On the other hand, the ability of ...cell types both promoters expressed in. The lab tested this application using one promoter to drive expression...experiments: While synthetic promoters with high rates of labeling are ideal of gene therapy applications...

...glycoprotein (BaEVRless) had high rates of transduction for most cell types they tested. Interested in using Nanoblades...technologies, Nanoblades had higher rates for editing efficiency and lower rates of off-target effects. Nanoblades... mouse bone marrow (BM) cells, primary human hepatocytes, and hematopoietic stem cells (HSCs) using Nanoblades...from GFP transgenic mouse  GFP  ~60-65% Human hepatocytes  Myd88  ~50% Human HSCs  Myd88  ~50% Table...dsDNA donor template. In vivo editing of mouse zygotes and mice with Nanoblades CRISPR has also been extensively...used to create transgenic mice by microinjecting zygotes with CRISPR components. However, this sometimes... to disrupt the tyrosinase (Tyr) gene in mouse zygotes, which results in albinism in mice. Of the eight...

... are light-inducible transcriptional effectors (LITEs) designed to bind specific genes and turn them on...reversible fashion. And that’s exactly what the LITEs now enable. In a Nature paper introducing the optogenetic..., Konermann and Zhang showed they could package LITEs in viral vectors and genetically target them to ... cell populations. They demonstrated use of the LITEs to control gene expression in mouse neurons and ...showed a rise in transcription in as little as 30 minutes after light stimulation. Under those optogenetic...Second, induction of transcription occurs within minutes of light exposure. Third, the response can be graded...players in normal biological processes and disease states. While LITE plasmids have clear application to ...

...cleavage at 1-12 off-target sites; in contrast, SpCas9 may cleave at ~90 sites, according to Kim et al. ...refresher on CRISPR/Cas9.  How was Cpf1 found and tested? Class 2 CRISPR systems, including the type II ...Again, they were in for a surprise! Whereas Cas9 generates blunt ends 3 nt upstream of the PAM site, Cpf1...activity in mammalian cells. From 16 diverse Cpf1 candidates, Zetsche et al. found two that display robust...homology-directed repair (HDR). Cpf1 also expands the number of sites that can be targeted by CRISPR to AT-rich regions...regions or AT-rich genomes that lack the 3’-NGG PAM sites favored by SpCas9. Since Cpf1 doesn’t require a ...confirmed. To prevent new editing post-HDR, repair templates should remove the PAM sequence. Comparing Cpf1...

...Addgene: And the BIL gates collection? Endy: The Boolean Integrase Logic (BIL) gates collection of parts...switches that enable cellular computing. These logic gates built from transcriptors are now available at Addgene...Addgene spoke with Endy more about the BIOFAB and BIL gates plasmid kits, the Biobricks Public Agreement, and...bioeconomy. Addgene: What do these BIOFAB and BIL gates kits – the first to be made available via Addgene...systems, plumbing, computers, and now through BIL gates we've implemented them in DNA. We thought these ...work, watch Endy's 10-minute YouTube primer on BIL gates.) Addgene: Why is it so important to you that these...as applied to products on the domestic market generates $300 billion dollars every year. That's two percent...

...off-target sites that they can later sequence for mutations. One method a researcher can use to test predicted...unbiased. Biased techniques only sequence certain sites in the genome predicted to contain off-target cleavage...techniques search the whole genome for off-target sites irrespective of in silico prediction. These techniques...computational methods. They identify possible off-target sites across the genome and pinpoint the location of mismatches...predicted off-target sites following a CRISPR experiment is targeted amplicon sequencing. The information ...low as .01% (Hendel et al. 2015). Low detection rates mean the investigator can be relatively certain ...information profile regarding putative off-target sites including both the edit rate and the repair product...

...resistance gene, NPTII, facilitates phosphorylation of kan, which inactivates it. The NPTII gene confers...recovery period after transformation (the dreaded 60 minutes shaking at 37 degrees) can be shortened since the.... As a result, a shorter recovery period of 30 minutes is possible with AmpR. So why even look at another...to produce satellite colonies when used on agar plates due to the fact that beta-lactamase is excreted...Cons: Prone to satellite colony formation on agar plates, less stable at high temperatures and in acidic... and is less susceptible to degradation in agar plates. This prevents satellite colonies from forming ...transformation recovery period than amp or carb (60 minutes). Pros: Widely available, cost-effective, confers...

... editors here! Testing and improving RNA editing from the Cas13b REPAIR system To test the robustness ...editor, the lab started with the Cas13 scaffold, testing a whopping 21 Cas13a orthologs, 15 Cas13b orthologs...and averages only ~50% RNA knockdown. In initial tests, Cas13b from Prevotella sep. P5-125 (PspCas13b) ...ClinVar database. They successfully edited 33/34 sites with a maximum of 28% editing efficiency in HEK293T...to fit into adeno-associated viral vectors, they tested truncated ADARDDs to see if they could shrink the...ADAR-RNA binding to decrease off-targets. Of the tested constructs, ADAR2DD(E488Q/T375G) (REPAIRv2) had...transcriptome-wide comparison, REPAIRv2 reduced off-target sites from the 18,385 observed with REPAIRv1 to merely...

...scientists using pLKO.1 variants learn about those updates? This is a problem many scientists struggle with...molecular cloning protocols, including making LB agar plates, restriction digest of plasmid DNA, and bacterial...is the creation of ZappyLabs. Currently in beta testing, the software aims to be a life science protocol...research labs to create their own lab or group websites. OWW is managed by BioBricks Foundation and is...detailed tutorials and easy to copy and modify templates (e.g. http://openwetware.org/wiki/Protocols/Template...Publishing Group. Like the previously mentioned sites, NPE allows scientists to join labs or groups and...Protocol Online is one of the early protocol sharing websites. It was created in 1999 and allows scientists ...