...variant 3' NGAG SpCas9 VQR variant  3' NGAN or NGNG  xCas9 3' NG, GAA, or GAT SpCas9-NG 3' NG ...SpCas9 3' NGG SpCas9 D1135E variant 3' NGG (reduced NAG binding) SpCas9 VRER variant 3' NGCG...CJ) 3' NNNNRYAC  Neisseria meningitidis (NM) 3' NNNNGATT  Streptococcus thermophilus (ST) 3' NNAGAAW...target sequence contained within the guide RNA and a 3-base pair sequence known as the Protospacer Adjacent...must contain a PAM sequence immediately downstream (3’) of the site targeted by the guide RNA. In the absence...researchers to target many different genomic loci. 3. Cleave target DNA resulting in a double-strand break...mediated DNA damage is a DSB within the target DNA ~3-4 nucleotides upstream of the PAM sequence. Cas9 Species...

...assist with restriction enzyme digestion (usually 3-6bp) Restriction Site: Your chosen restriction site...Primer will use the sequence 5'-ATGTGGCATATCTCGAAGTAC-3' for the region that binds the ORF and we will add...our Forward Primer 5'-GAATTCATGTGGCATATCTCGAAGTAC-3'. Many restriction enzymes do not cut DNA efficiently...more information). Thus, we recommend that you add 3-6 bases upstream of your restriction site to improve... sequence of 5'-TAAGCAGAATTCATGTGGCATATCTCGAAGTAC-3'. For the Reverse Primer, the design is similar, but...including the stop codon (5'-TGGCATATCTCGAAGTACTGA-3'), then adding NotI (GCGGCCGC) and then TAAGCA to ...sequence of 5'-TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3' (30bp with 18bp of homology to the ORF). We now need...

...polymerase leaves a single deoxyadenosine (A) at the 3' ends of PCR products. The complimentary “T” in the...this enzyme recognizes the DNA sequence 5´-(C/T)CCTT-3' and digests double stranded DNA at this sequence.... is stored as a covalent bond between the cleaved 3′ DNA strand and a tyrosyl residue of topoisomerase... provide vectors or cloning arms with overhanging 3´ deoxythymidine (T) residues that are covalently linked...potential positive clones. Once you introduce your 3’-end “A” overhang insert, the magic of TOPO cloning...Reaction: Mix together the PCR product and TOPO Vector.  3. Incubate 5 Minutes at Room Temperature: You can place...proofreading polymerases will also remove all unpaired 3’ ends in your PCR product. If you need to decrease...

...ligase 25 °C 1 hour 3-base or 4-base T4 DNA ligase 25 °C 18 hours 3-base or 4-base T4 ...ligase 37 °C 1 hour 3-base or 4-base T4 DNA ligase 37 °C 18 hours 3-base or 4-base Bbsl-HF...-16 °C cycling 3-base Sapl, 1x NEBridge Ligase MM 37-16 °C cycling 3-base Table 1: Enzyme... the same overhang twice; (2) avoid palindromes; (3) no overhangs with the same three nucleotides in a...could be done using overhang sets that broke rules 3—5. Fidelity, it turns out, might depend more on the...must be used. Each of the tools works with either 3-base or 4-base overhangs, and with a variety of different...BspQI, 1x NEBridge Ligase MM 42-16 °C cycling 3-base  PaqCl®, 1x T4 DNA Ligase Buffer 37-16 ...

...Figure 3: BaF3 cells were transduced with H2B-FT virus and grown in varying concentrations of IL-3. Cells..., which depend on IL-3 to proliferate. The cells cultured with a low dose of IL-3 showed reduced proliferation...high IL-3 showed mostly blue fluorescence, indicating a quick cell cycle. Cells that had IL-3 withdrawn...Cells grown in high concentrations of IL-3 show a high proportion of blue fluorescence, indicating cell ...proliferation. BaF3 cells maintained in media without IL-3 show only red fluorescence. Image from Eastman et ...Streptococcus canis Cas9 nuclease that has a broad 5′-NNG-3′ PAM. Get the plasmids at Addgene The Human GlycoGene... plasmids you need. If you'd ever like to write about a recent plasmid deposit please sign up here.  Here's...

...the PITCh vector should be constructed with 5’ and 3’ microhomology to the insertion locus flanking the...the GFP-Puro cassette and one in between the 5’ and 3’ microhomologies in the genomic locus. The first two...breaks allow for two sets of microhomologies (5’ and 3’) to anneal, knocking the GFP-Puro cassette into the...plasmid contains a GFP-Puro cassette flanked by 5' and 3' microhomology and PITCh-gRNA binding sites. This ...microhomologies in the PITCh vector ~20 bp 5’ and 3’ microhomologies are added to the GFP-Puro cassette...a vector containing Cas9 and the PITCh-gRNA. Step 3: Contransfect the PITCh vector with the vector carrying...of clones displayed proper insertion at the 5’ and 3’ junctions, respectively. These results indicate that...

...bigger than ∼3 kb are packaged less efficiently. Subcomponents: U3; Unique 3'; region at the 3' end of viral...SIN γ-retrovirus lack most of the U3 region of the 3’ LTR and are primarily used in gene therapy research...viral genomic RNA (but found at both the 5' and 3' ends of the provirus). Contains sequences necessary...transcription. R; Repeat region found within both the 5' and 3' LTRs of retro/lentiviral plasmids. U5; Unique 5';...the viral genomic RNA (but found at both the 5' and 3' ends of the provirus). 5’ LTR Acts as an RNA...proceeds through U5 and the rest of the provirus. 3’ LTR Terminates transcription started by 5' LTR ...packaging methods. The three components described above (envelope, packaging, and transfer) are supplied...

...genetic tools facilitates plant science. Challenge #3: Intellectual property There is another challenge ...Revolution Bioengineering. Read their previous blog post about how they started their company here. The first transgenic... foundation of life on earth, and opportunities abound to build better fuels, feeds, foods, and fibers... we hope to inspire and open a new conversation about GMOs.  Challenge #2: Technical obstacles to plant...not understand, and the complicated and unique metabolic processes of plants are being investigated on ...biochemical pathways involved in central anthocyanin metabolism are well understood. Additional work has also...knockouts to better understand developmental, metabolic, and photosynthetic processes. Often the tools...

...market; you need one with 5’->3’ polymerase activity (for amplification), 3’->5’ exonuclease activity (increases...on either side of the desired mutation (usually 1-3 mismatched bases) is sufficient for your primers to...present, a minor extension can usually ensure that the 3’-base(s) do not form secondary structures. The introduction...template (e.g. 0.1-1.0 ng/μl). Smaller plasmids (~3 kb) are generally more efficiently amplified than ...addition of DMSO to the PCR reaction (usually around 3% final concentration). The DMSO reduces secondary ...(increases amplification fidelity), and no 5’->3’ exonuclease activity (which could potentially truncate... TA-cloning). Such non-complementary bases at the 3’-end interfere with plasmid reconstitution, and consequently...

... (Nishimasu et al., 2014). Figure 3: pegRNAs whose 3′ extension begins with a C base can cause...Created with BioRender.com. The first base of the 3′ extension of the pegRNA should not be C. A C base...Add silent mutations near point mutations to create 3-base (or longer) tracts of edited bases. DNA mismatch...targets single-base mismatches (left), but “bubbles” of 3 or more mismatched bases can often evade MMR (right...epegRNAs: epegRNAs include structured motifs at their 3′ end to protect them from degradation. When designing...protein La to protect the end of the pegRNA. Adding 3′ polyU tracts to the end of pegRNAs (but not epegRNAs...prime editing efficiency. Nature Biotechnology, 40(3), 402–410. https://doi.org/10.1038/s41587-021-01039...

...overlap between them (Table 1). In each construct, 3 copies of the fluorescent protein were fused in-frame...from the tyrosine-protein kinase Lyn) Membranes 3 mCherry 587nm/ 610 nm human β-Actin Actin 4 Citrine...sites of the MCS are only regenerated at the 5’ and 3’ termini of the ligation product, and the restriction... them, but rather, can only be added to the 5’ or 3’ terminus using the newly generated MCS. MXS-chaining...the third module is added to the 5’ terminus or the 3’ terminus of the ligation product. Comparison to other...chaining-based cloning methods are listed in Table 3. The goal of high throughput cloning is to facilitate...Chaining by emailing us at [email protected]. Table 3: Chaining Based Cloning Methods Method Restriction...

...hype. 3. “Passion gives you energy.” What is your passion? Even if no one else is excited about it, distinguish...success? We asked leading professors for their top 3 tips. Tom Ellis 1. Keep up with the literature. ... that your experiment is definitely not a fluke.” 3. Engage in various projects and help colleagues with... competitive. Be curious and interdisciplinary.   3. “There are many different ways to succeed – as loner...: Read the Newest Posts in this Blog Series   Our 3 PIs answer more questions, including: Advice for ... passionate about. Check out my previous post if you're looking for more information about the benefits...every spurious result under the sun. Academia is about convincing results, and nothing is more convincing...

...information about the aims and approach of CeNGEN, check out their publication.   3’UTRome The 3’UTRs of ... the role of 3’UTRs, the Arizona State University (ASU) Biodesign institute developed a 3’UTR database...This database provides detailed information about C. elegans 3’UTRs structures, alternative polyadenylation...Mangone, Marco, et al. "UTRome. org: a platform for 3′ UTR biology in C. elegans." Nucleic acids research...unc- 119 + selection." MicroPublication 1 (2018): 2–3. Wang, Han, et al. "cGAL, a temperature-robust GAL4...resources on the Addgene blog Learn more about SapTrap Read about CRISPR/Cas9 in C. elegans Browse our Plasmids...always emphasized the need for open science and collaboration. The field already has comprehensive reference...

...scientific Twitter list were excluded from the dataset. 3. 60% of scientists on Twitter are male and 40% are...with scientific domains <10% of the time. Figure 3: Top 20 Domains Tweeted by Scientists. 6. The top ...use Twitter professionally to network, learn more about alternative careers for scientists, and share cool....” This paper addresses the following questions about scientists on Twitter: who are they? What do they...

...2010 3 Nature  3.3% 10.5%   3.2  9.6 2 2010 167  Nature 3.3%  10.5%  3.2  534.1 3 2010 ...plasmids in our data were published in Nature, but only 3 percent of papers without Addgene plasmids were. Thus...journal-adjusted citation measure we re-weight the 3 percent of Nature papers without Addgene plasmids ...2010 3 Applied and Environmental Microbiology  1.6%  1.2% 0.75   2.3 4 2010 167  Applied and Environmental...deposited at Addgene still accrue on average 2 to 3 times as many citations as other papers in the same... with plasmids deposited at Addgene – typically about four times as many as papers without plasmids deposited...

...preferred PAM is 5’-TTN, differing from that of Cas9 (3’-NGG) in both distribution and GC-content, which might.... Additionally, whereas Cas9 generates blunt ends 3 nt upstream of the PAM site, Cpf1 cleaves in a staggered...Cpf1 activity, except when they were present in the 3’ end of the target sequence (bases 19-23). Cpf1 is...gRNA positions 1, 8, 9, and 19-23. Accordingly, the 3’ end of the gRNA target sequence does not have an ...decrease in Cpf1 activity with 4-6 base deletions at the 3’ end of the target sequence. Cpf1’s strength may lie... Find plasmids from this publication at Addgene. 3. Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker...System. Cell. 2015 Sep 23. pii: S0092-8674(15)01200-3. doi: 10.1016/j.cell.2015.09.038. PubMed PMID: 26422227...

...engineering has come. For comparison, in 2007 it took about 3 months to knockout a single allele in a human line...thing ask incredulously, “You seriously didn’t add 3 microliters of 5 mM star anise?” This is something...expression was across the different cells (Figure 3).   Figure 2: Cas9 expression in monoclonal...stable cell pool were then expanded for an additional 3 weeks under blasticidin selection. Anti-Cas9 Western... control (WT). Vinculin is a loading control. 3. In terms of making the monoclonal line: I had never...with many additional tips can be found here. Figure 3: A surprise for the author. Checking Cas9 expression...would still be able to identify the cleavage product. 3. The Surveyor kit had a lot of good tips for first...

...preferred PAM is 5’-TTN, differing from that of Cas9 (3’-NGG) in both genomic location and GC-content. After...for a surprise! Whereas Cas9 generates blunt ends 3 nt upstream of the PAM site, Cpf1 cleaves in a staggered... AT-rich regions or AT-rich genomes that lack the 3’-NGG PAM sites favored by SpCas9. Since Cpf1 doesn...Cpf1 activity, except when they were present in the 3’ end of the target sequence (bases 19-23). Cpf1 is...gRNA positions 1, 8, 9, and 19-23. Accordingly, the 3’ end of the gRNA target sequence does not have an ...decrease in Cpf1 activity with 4-6 base deletions at the 3’ end of the target sequence. Figure 2: Effects... Find plasmids from this publication at Addgene. 3. Kim, Daesik, et al. Genome-wide analysis reveals ...

...presence of additional helper sequences (2-3). As alluded to above, termination and polyadenylation of Polymerase...transcribed, and typically occur directly after any 3’ regulatory elements, such as the polyadenylation ...is non-specific and can be added to any accessible 3' end. The presence of the poly(A) tail targets the...releases the mRNA from the polymerase and creates a free 3' end which is now available for polyadenylation. The...Landick R. Bacterial Transcription Terminators: The RNA 3′-End Chronicles. Journal of molecular biology. 2011...are required for efficient rabbit beta-globin mRNA 3' end formation. Cell. 1987. PMID: 3568131.  Hager ...how the Promoter Controls Gene Expression Learn about New Cloning Techniques Read all of our Plasmids ...

...TRICK system, the 3’ UTR of the reporter mRNA is labeled by PCP-mCherry (Figure 2). The 3’ UTR also contains...protein (NLS-PCP-GFP) an RFP protein that binds to the 3’ UTR of the reporter mRNA via an MS2 coat protein ...multiple hairpin sequences into the coding sequence and 3’ UTR. Before beginning experiments, it’s important...PMC4451088 Find plasmids from this paper at Addgene. 3. Yan, Xiaowei, et al. “Dynamics of Translation of ...Additional Resources the Addgene Blog Learn More about SunTag Catch up on Your GFP Background Choose the...