...found climbing mountains or playing with his two cats. Keira Havens grew up in Hawaii where she was fascinated...genetic tools facilitates plant science. Challenge #3: Intellectual property There is another challenge ...biotechnologists face is public perception of genetic modification (GM) and synthetic biology. The mistrust and...cannot build what we do not understand, and the complicated and unique metabolic processes of plants are ...

...editing (Fig. 2 and 3) (5, 3). Benefits of truncated gRNAs Importantly, the use of truncated guides has largely...its nucleolytic activity is eliminated (Fig. 1) (1, 3). The gRNA can be further modified by incorporation...methods 12.9 (2015): 823-826. PubMed PMID: 26167643. 3. Kiani, Samira, et al. "Cas9 gRNA engineering for ...protospacer, in the genome. Native Cas9 has two catalytic domains, each of which cleaves one strand of DNA... addition to its utility in making targeted modifications to DNA, Cas9 can be reprogrammed to serve as...as a regulator of gene expression. Its catalytic domains can be mutated to inactivate the protein’s nucleolytic...target sites. Regulating gene expression with truncated gRNAs An alternative approach is to modulate Cas9...

...Forward Primer will use the sequence 5'-ATGTGGCATATCTCGAAGTAC-3' for the region that binds the ORF and ...primer, making our Forward Primer 5'-GAATTCATGTGGCATATCTCGAAGTAC-3'. Many restriction enzymes do not cut...final Forward Primer sequence of 5'-TAAGCAGAATTCATGTGGCATATCTCGAAGTAC-3'. For the Reverse Primer, the design... the ORF, including the stop codon (5'-TGGCATATCTCGAAGTACTGA-3'), then adding NotI (GCGGCCGC) and then... This gives us a sequence of 5'-TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3' (30bp with 18bp of homology to ...chose for our reverse primer (5’-TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3’) into this calculator we get a ...assist with restriction enzyme digestion (usually 3-6bp) Restriction Site: Your chosen restriction site...

... for the amplification step since Taq polymerase leaves a single deoxyadenosine (A) at the 3' ends of ...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...

...high IL-3 showed mostly blue fluorescence, indicating a quick cell cycle. Cells that had IL-3 withdrawn...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... concentrations of IL-3 show a high proportion of blue fluorescence, indicating cell proliferation. BaF3...BaF3 cells maintained in media without IL-3 show only red fluorescence. Image from Eastman et al., 2020...Streptococcus canis Cas9 nuclease that has a broad 5′-NNG-3′ PAM. Get the plasmids at Addgene The Human GlycoGene... labeling proteins with different subcellular locations. Image from Goudeau et al., 2020. Goudeau...

...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 ...

...market; you need one with 5’->3’ polymerase activity (for amplification), 3’->5’ exonuclease activity (increases...increases amplification fidelity), and no 5’->3’ exonuclease activity (which could potentially truncate the ...on a high percentage agarose gel (~3%). Note that primer duplication will evade detection in the initial...PubMed PMID: 8470801. 3. Saiki, R. K., et al. "Primer-directed enzymatic amplification of DNA." Science 239...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 ...

...proper insertion at the 5’ and 3’ junctions, respectively. These results indicate that PITCh is a robust method...PMID: 26456817. Find plasmids from this publication at Addgene. 3. Zhang, Chi, Xiuhua Meng, Xiaolei Wei...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...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...

...Teacher, Vol. 79 No. 3, March 2017; (pp. 208-223). Link: http://abt.ucpress.edu/content/79/3/208 2. Fire, S....Nature, 391 (1998), pp. 806-811. PubMed PMID: 9486653. 3. Newmark, Phillip A., et al. "Ingestion of bacterially...DE3. For Phagocata morgani and Phagocata gracillis, the process is a little more complicated. These strains...Girardia sp., Dugesia dorotocephala, Phagocata morgani, and Phagocata gracillis. There are 11 different ... coli. Thus, to get the dsRNA into Phagocata morgani and Phagocata gracillis, one has to purify the dsRNA... in situ hybridization experiments. A recent publication [1] from the Sánchez lab describes the plasmids...the deposit) are described in the Sánchez lab publication [1] so that the students performing these experiments...

...come in contact with target cells (Fig. 3).     Figure 3: The life cycle of a retrovirus. Image...protein (IN) during step 3 (King et al., 2008) (Hu & Temin, 1990). At step 3, the double stranded DNA ...Image courtesy of Jeremy Luban, MD. Figure 3 depicts the viral life cycle. One should ponder this figure...of DNA into the host cell’s genome (shown in step 3), but note also that it is worthwhile to generalize...complex retroviruses. Microbiological Reviews, 56(3), 375–394. https://doi.org/10.1128/MR.56.3.375-394.1992...produce infectious murine leukemia virus. Virology, 46(3), 866–876. https://doi.org/10.1016/0042-6822(71)90087...retroviruses ” (Cullen, 1992) because of their more complicated genomes. Here, we will mostly use the gamma-retroviruses...

...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...

...approach of CeNGEN, check out their publication.   3’UTRome The 3’UTRs of mRNA are important for post-... the role of 3’UTRs, the Arizona State University (ASU) Biodesign institute developed a 3’UTR database...mutations with unc- 119 + selection." MicroPublication 1 (2018): 2–3. Wang, Han, et al. "cGAL, a temperature-robust...database provides detailed information about C. elegans 3’UTRs structures, alternative polyadenylation for all...Mangone, Marco, et al. "UTRome. org: a platform for 3′ UTR biology in C. elegans." Nucleic acids research...the OpenWorm website. microPublication microPublication is a unique publication for the worm community...into larger publications and would otherwise be lost in lab notebooks forever. microPublications are peer-reviewed...

...PMID: 27347757. Find plasmids from this publication at Addgene. 3. Zetsche B, Gootenberg JS, Abudayyeh ...01200-3. doi: 10.1016/j.cell.2015.09.038. PubMed PMID: 26422227 Find plasmids from this publication at ...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...

...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...databases available, Web of Science. For each publication, Web of Science provides basic bibliographic ...Science, PLOS One, and PNAS. We then broke the publications from these journals into two groups: those containing...

...-TTN, differing from that of Cas9 (3’-NGG) in both genomic location and GC-content. After sequencing and...PMID: 27918548. Find plasmids from this publication at Addgene. 3. Kim, Daesik, et al. Genome-wide analysis...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. Cpf1’s strength may ...

...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 ... While ribosome profiling co-opts the physical location of the ribosome, TRICK (Translating RNA Imaging...development, the osk-TRICK reporter was double labeled, indicating translational repression. During later stages...ribosomes after initiation, to calculate a ribosome translocation rate of ~3.5 codons/second. They also explored...

...spending time with her horse, and wondering what her cat is thinking. Find her on Twitter @JessicaSacher. ... step involves digesting phage genomic DNA into 1-3 kb (approximately gene-sized) fragments so that these...bulk of resultant fragments falling between 1 and 3 kb results in optimal likelihood of capturing a whole...filter paper and saturate each with lysis solution (~3 ml each), pouring off any excess. When your protein...Expression Vectors in the FX Cloning System Kit  Step 3: Probe random phage DNA fragments with live host cells...PMID: 27536293. PubMed Central PMCID: PMC4971087. 3. Simpson, David J., Jessica C. Sacher, and Christine..." Chemical Reviews (2016). PubMed PMID: 27319741. 3. Simpson, David J., Jessica C. Sacher, and Christine...

...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...to the presence of additional helper sequences (2-3). As alluded to above, termination and polyadenylation...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 ...type are generally broken down into two broad categories, prokaryotic or eukaryotic, based on the functional...

...Gutless AdV can be found in reference 3. Production, amplification, and quality control of 2nd generation...size, and consists of an icosahedral capsid made of 3 types of proteins: fiber, penton and hexon proteins...sequenced to confirm its integrity. Initial production (2-3 weeks) - Here you’ll produce the primary recombinant... will turn yellow. Do NOT change the media (add 2-3 mL of fresh media once a week), and do NOT harvest...infected cells will become round and clump together ~3 days post infection. The amplified virus can be harvested...al. A practical guide for using the AdEasy System. 3. Jager, Lorenz, et al. "A rapid protocol for construction...Each round of amplification should result in a 10-100-fold increase in virus. Purification (2 days) - Purification...

...between deletion size and frequency [3]. There are several applications in which the production of deletions...to deletions following paired DSBs is inversions [3]. These rearranged segments may also be informative...is likely to bear an indel or inversion mutation [3]. In the CRISPR era, the genomic landscape may be ...level. Science 2013 Oct 11;342(6155):253-257. PubMed. 3. Canver MC, Bauer DE, Dass A, Yien YY, Chung J, Masuda... of Visualized Experiments (JOVE). The video publication by Stuart Orkin and Daniel Bauer's labs details...useful to study haploinsufficiency. Subsequent modifications at the remaining allele within the deletion ...