...cutting enzymes: NotI at the 5' end and AscI at the 3' end. These inserts can then be sub-cloned into a ...benchmarked protein microarray-based platform for the identification of novel low-affinity extracellular protein...

...Bacteria SpeI + HindIII none S. pyogenes Qi pDD162 (Peft-3::Cas9 + Empty sgRNA) 47549 C. elegans yes, cut S. ... Puro Abbosh pCFD3-MS2tail-Scaffold 78906 Fly dU6:3 none S. pyogenes Virmilion Perrimon pHDE-35S-Cas9-...polycistronic lentiviruses. Can express Cas9 with up to 3 gRNAs. Frew pSQT1313 Mammalian Single plasmid for ...expression of two gRNAs from Drosophila U6:1 and U6:3 promoters. Bullock pCFD5 Drosophila Utilizes endogenous...RNA Targeting RNA Targeting RNA Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled...

...Fishell 83898-AAV1 pAAV-mDlx-ChR2-mCherry-Fishell-3 ChR2-mCherry fusion expression in forebrain GABA-ergic...Fishell 83898-AAV9 pAAV-mDlx-ChR2-mCherry-Fishell-3 ChR2-mCherry fusion expression in forebrain GABA-ergic...Fishell 83898-AAVrg pAAV-mDlx-ChR2-mCherry-Fishell-3 ChR2-mCherry fusion expression in forebrain GABA-ergic... the human brain through the development and application of innovative tools enabling large-scale real-time...or when BRAIN Initiative grants are noted in publications associated with Addgene materials. Plasmids ...Gradinaru 105448-AAV9 pAAV-hSyn-DIO-ChrimsonR-mRuby2-ST Cation channelrhodopsin ChrimsonR fused to mRuby2 fluorophore...108912-AAV9 pAAV-CAG-DIO-ChroME-ST-P2A-H2B-mRuby3 Cation channelrhodopsin ChroME targeted to the neuronal...

...Combinatorial Strategies Connie Cepko 2:45 PM Break 3:00 PM (Group Discussion) Technologies and Resources...contribute to this publication. Citation information for openly shared communication: Adeno-Associated ...Addgene's 2018 AAV Education and Development (AAVED) workshop series. The... The AAV Education and Development ...Details Date: Friday September 21, 2018, 9 AM - 5 PM Location: Addgene, 75 Sidney St, Cambridge, MA, 02139 Organizing...useful to help improve this technology or its application? What are some current obstacles that researchers...prepare and compile resources for this meeting's publication, we are asking participants to share practical...

...Cre lac Bacterial Ekker 47551 pDD104 (Peft-3::Cre) Cre eft-3 C. elegans Goldstein 48201 CAG-GFP-IRES-CRE...deltaG)-Cre Cre CMV RABV Jessell 73717 pMLS328 Cre eft-3 C. elegans Jorgensen 75267 pAAV-CIBN-CreC photo-activatable...Vivo Electroporation. Proc Natl Acad Sci. Jan 16;104(3):1027-32. doi: 10.1073/pnas.0610155104. Epub 2007 ... maintained). Translocation: If the sites are on separate DNA molecules, a translocation event is generated...recognizes 34 bp loxP sites, and the orientation and location of the loxP sites determines how the genetic material... of rearrangements: inversion, deletion and translocation. Inversion: If the loxP sites are on the same...present. By expressing Cre at specific times or locations, you can precisely control expression of your ...

...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 ...

...genetic model organism due to their rapid life cycle (3 days from egg to adult) and easy lab maintenance. ...Efficient Marker-Free Recovery of Custom Genetic Modifications with CRISPR/Cas9 in Caenorhabditis elegans. ...consortium of biologists and computer scientists dedicated to providing the research community with accurate...

...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...

... rpoZ- on plates lacking histidine and containing 3-AT Scot Wolfe Bacterial one-hybrid system for testing...promotes growth on plates lacking His and containing 3-AT. FLIParaF.Ec-250n 18855 N/A FRET between CFP and...Genome Engineering Visualization and Tagging Purification Controlled Expression Reporter Plasmids Reporter...Reporter Plasmids Tagging and Visualization Purification Controlled Expression Reporter Plasmids You may...Bacteria? E. coli are commonly used to store and replicate plasmids of all types, but beyond that, researchers...sites within this plasmid. Return to Top Protein Purification Sometimes looking at a protein inside cells ...and, epitope tags can be used to simplify the purification process. Using plasmids in this collection, ...

...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...

... new vial of early passage cells. Approximately 2-3 weeks post-thaw, cell culture supernatant is tested...minimized by using an endotoxin-free plasmid purification protocol. To minimize the immunogenic properties...posted on the material page for the corresponding catalog item (see maps section for images). AAV Pro cells...typically prepared using endotoxin-free plasmid purification kits. Resources Browse lentiviral preps available...

...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...