...protocols that recommend eluting in water, Tris-EDTA (TE), just Tris buffer, or some other variations. Does...experiments you can use your plasmid.  DNA elution choices: TE, Tris buffer, or water Figure 2: Tris-EDTA,...choose could affect your downstream experiment. TE (10 mM Tris-HCl,1 mM EDTA, pH 8.0) buffer is the ...In the situation where you need to use plasmid in TE for any enzymatic reaction, an effective possibility...adenine or guanine) from the DNA. These apurinic sites if in double stranded DNA are repaired by base excision...time. Tris buffer controls the pH, while the EDTA chelates any divalent cations like Mg2+, preventing the...

...digestion and PCR products with sterile water (instead of TE buffer) to ensure optimal salt concentrations in ...room temperature for 30 minutes. Inactivate T4 Pol by heating to 75° for 20 minutes. Volume (μl) Reagent... for 5 minutes at room temperature, then add 1 μl of 25 mM EDTA, followed by another 5 minutes at room...Protocols Overview Ligation Independent Cloning (LIC) obviates the need for the time-consuming ligation step ...with a single enzyme, and remove the restriction sites from the final product (no "cloning scars"). A ...reaction may be stopped by heating to 75° for 20 minutes. Step 6: Anneal and Transform Mix your treated ...

...recognizes 34 bp loxP sites, and the orientation and location of the loxP sites determines how the genetic...region of DNA between the loxP sites is reversed. Deletion: If the sites face in the same direction, the...If the sites are on separate DNA molecules, a translocation event is generated at the loxP sites. For a... that recognize specific mutant loxP sites, VloxP and SloxP sites respectively. Split Cre: Cre recombinase...injection into fertilized zygotes Mammalian Sauer 21270 pBigT Cre activates gene of interest by removing... 38042 pAAV-CA-FLEX Cre activates gene of interest, using 4 mutated lox sites AAV Uchida Cre Reporters...expression. Flp recombinase recognizes FRT sites. Flp binding to these sites can result in excision, inversion ...

...acetyllysine reader BRD3R promotes human nuclear reprogramming and regulates mitosis. Nat Commun. 2016...and c-Myc from polycistronic cassettes KLF4 N-terminal variance modulates induced reprogramming to pluripotency...30. Zhou Astrocytes Dopaminergic Neurons Lentiviral Human Efficient conversion of astrocytes to functional...Fibroblasts Astrocytes Lentiviral Mouse Direct conversion of fibroblasts into functional astrocytes by defined...Oct4, Sox2, Klf4, and c-Myc from polycistronic cassettes. piggyBac transposition reprograms fibroblasts...To Delivery Method Species Article PI iPSCs Cardiomyocytes Lentiviral Human Robust cardiomyocyte differentiation...Sci U S A. 2012 May 29. Palecek Fibroblasts Cardiomyocytes Lentiviral Mouse Optimization of direct fibroblast...

...MS2 binding sites within this plasmid. pZE31-DsRed-ms2x2 26248 MS2 coat protein binding sites Philippe Cluzel...strain USO hisB- pyrF- rpoZ- , this promotes growth on plates lacking His and containing 3-AT. FLIParaF....microscopy. pZE31ms2 25860 MS2 coat protein binding sites Philippe Cluzel RNA localization: Contains a 2x ...appropriate cDNA has been fused to the MS2 binding sites within this plasmid. Return to Top Protein Purification...N-terminal GST tag and TEV cleavage site. Includes sites for LIC cloning, and a "stuffer" fragment that includes...mOCR, MBP, with and without SUMO or TEV cleavage sites to remove tags Scott Gradia Arabinose inducible ...MBP, GFP, with and without SUMO or TEV cleavage sites to remove tags. Scott Gradia Lactose/IPTG inducible...

...Fusions; Coding Prefix alleviates RBS-CDS issues. Prohibited Restriction Sites: EcoRI, XbaI, SpeI, PstI...genome. Prohibited Restriction Sites: EcoRI, SpeI, NheI, PstI Restriction Sites to Avoid: PvuII, XhoI, AvrII...assembly standards – combinations of restriction sites which preserve a standard prefix and suffix sequence... are added. Therefore, the same reaction which ligates a promoter into an empty backbone can be repeated...restriction enzymes used for assembly are the prohibited sites and must be omitted from the coding sequence of ...backbone. For some standards, there are additional sites which should be avoided whenever possible. Other...PstI Restriction Sites to Avoid: NotI For more info, visit iGEM: BioBrick (RFC 10) Back to Top BioBrick...

...2013). We also have an older system using two cassettes to express the CRISPR RNA array and tracrRNA separately...guide RNA: These plasmids contain two expression cassettes, a human codon-optimized SpCas9 or SpCas9n, and...tracrRNA: This plasmid contains three expression cassettes. To target a given site, the plasmid can be digested...loops at tetraloop and stemloop - Contains BbsI sites for insertion of guide sequences Addgene plasmids... and EF1a-zeo resistance marker. Contains BsmBI sites for insertion of guide sequences Zhang Lab SAM Cloning...guide RNA: These plasmids contain two expression cassettes, a human codon-optimized SaCas9 driven by either...AAV plasmid for sgRNA cloning. GFP-KASH fusion facilitates FACS sorting of cells and nuclei. Return to top...

...D'Ippolito AM, Vockley CM, Thakore PI, Crawford GE, Reddy TE, Gersbach CA. Nat Biotechnol . 33(5):510-7. PMID:...have additional sites throughout the genome where partial homology exists. These sites are called off-...partial homology to additional sites throughout the genome. These sites are called off-targets and should...efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing. 2015....modifications to Cas9. As discussed previously, Cas9 generates double-strand breaks (DSBs) through the combined...mutant of SpCas9, retains one nuclease domain and generates a DNA nick rather than a DSB. Thus, two nickases...preventing strand separation and cutting at off-target sites. Similarly, SpCas9-HF1 lowers off-target editing...

...increasing on-target editing rates and decreasing off-target editing rates. Advances in this area include...offering flexibility and easy multiplexing/scaling. Prokaryotes have long utilized CRISPR as a powerful defensive...elucidated until 2007. Scientists hypothesized that prokaryotes used CRISPR as part of an adaptive immune system...would bind. (5) After the complex binds, Cas9 separates the double stranded DNA target and cleaves both...seen in human patients, protein tags, or loxP/FRT sites, among others. Homology-directed repair (HDR) allows...packaged in AAV. Cas9 orthologs also have distinct PAM sites that do not cross react, allowing multiple Cas9s...engineered mutant Cas enzymes with alternative PAM sites . Cpf1 , another CRISPR nuclease, recognizes a 5...

...response has developed, the antibodies are collected, tested against the antigen, packaged, and sold. This is...immortalized B cells). The genetic reorganization that creates variety in the antibody allowing for their extreme...class therefore has a valency, or number of binding sites available per antibody unit, which is typically ...multiplied by two, as each antibody has two binding sites. Isotype Description Configuration and Valency Subclasses...Monomer with a valency of 2 IgE Protects against parasites and is responsible for driving allergic reactions...in the lab. Check for and record any expiration dates. Buffers Buffers are a concern for conjugating and...reaction in the presence of several different substrates. The luminescence produced by the reaction can...

... mTORC1 integrates these signals to determine the nutritional state of the cell and regulates the switch...signaling of mTOR, a key metabolic regulator, correlates with poor prognosis in many cancers. Cancer...anabolic to catabolic processes. Active mTORC1 promotes protein and lipid synthesis, and increases energy...Enhanced signaling through either mTORC1 or mTORC2 correlates with poor prognosis in many cancers, including... cancer. Loss of the tumor suppressor p53 also promotes aberrant mTORC1 activity, and multiple familial...mTORC1. The hyperactivation of mTORC1 in cancer promotes the conditions needed for uncontrolled cell growth... which appears to signal upstream of Akt, also promotes cell survival and proliferation. mTOR Pathway ...

...nuclease sites, this tool from the Joung Lab has been expanded to identify potential DNA target sites for ...access tool from Synthego allows you to determine rates of CRISPR editing at a given locus. To use the tool...sequence, functional noncoding elements and off-target sites. This algorithm allows for the quantification of... splice site mutations. The CRISPResso suite accommodates single or pooled amplicon deep sequencing, WGS...Church Lab : a tool that identifies putative target sites for S. pyogenes Cas9, S. thermophilus Cas9, or Cpf...probability of off-target effects. Cas-Designer integrates information from the Kim Lab's Cas-OFFinder and...online predictor Identifies candidate sgRNA target sites by off-target quality. Validated for gene inactivation...

...ΔA all TAG sites changes to UAG, RF1 function removed George Church 48999 C321 all TAG sites changes to...all TAG sites changes to UAG, RF1 function removed Farren Isaacs 87359 C321.∆A.opt all TAG sites changes...to UAG George Church 49018 C321.ΔA.exp all TAG sites changes to UAG, RF1 function removed, MutS restored...mutation rate George Church 68306 C321.ΔA all TAG sites changes to UAG, RF1 function removed, deletion of...Strain 192872. Jesse Rinehart 69493 MCJ.559 TAG sites, RF1 function removed, genomic deletions for improved...incorporation Michael Jewett 69495 rEc.13.delA 13 amber sites changed to TAA, RF1 function removed Michael Jewett.... George Church 98564 C321.Ub-UAG-sfGFP all TAG sites changes to UAG, RF1 function removed, with Ubiquitin-UAG-sfGFP...

...Phosphopeptide Library ). The phosphorylation sites (“phosphosites”) consist of a central phosphoserine site...expressing recombinant phosphoproteins. These attributes make rEcoli XpS ( 192872 ) and pSerOTS-C1* ( ...site-specific incorporation of phosphoserine in >36,000 phosphosites by tandem mass spectrometry (Barber et al, Nat...synthesized using supD tRNA ( 68307 ) to generate the phosphosites with serine instead of phosphoserine (i.e. a... can then be used to identify candidate human substrates of kinases of interest by mass spectrometry in...library ( 188536 ) into 10 subpools of ~11,000 phosphosites ( 188526 ; 188527 ; 188528 ; 188529 ; 188530...precise knowledge of phosphorylated positions for testing phosphoproteomics methods, proteomics search algorithms...

...system for eukaryotes. At the time, bacterial systems from E. coli presented the best candidates for inducible...were tested for functionality in mammalian cells. The lac system with the IPTG inducer was tested first...induction. In 1992, Manfred Gossen and Hermann Bujard tested the tet system in a mammalian cell system (HeLa...these tetO sequences and the activation domain promotes expression. In the presence of tetracycline, tetracycline...26803 pEnt L1L3 EF1a-tTA-2 Contains Gateway L1L3 sites and EF1α promoter driving tTA; contains insulator...cell transcription factor fusion derivatives, regulates inducible gene expression in mammalian cells. ...

...near-matches to reduce risk of cleavage away from target sites (off-target effects). Ensure that the guide sequences...primers upstream and downstream of the sgRNA cleavage sites (“deletion band”; Figures 1 - 2 ). In the absence...sorted cells individually into 96-well round-bottom plates using sorter or by using limiting dilution at 30...conditions for each primer pair designed based on testing the bulk sorted cells. Run samples on 2% agarose... per well for a final volume of 150 μl. This facilitates screening by allowing a multichannel pipette ...100 μl each into two separate 96-well flat-bottom plates. Keep one plate to allow for clones to grow and... into DH5α E. coli cells and plate onto LB agar plates with the relevant antibiotic. Select multiple colonies...

...functionally distinct interneurons in mice, non-human primates and humans. Nat Neurosci. 2020 Dec;23(12):1629...Plasmids Rylan Larsen Calcium Calcium sensor in astrocytes (GCaMP6f), cytosolic Conditions and constraints...413-29. Baljit Khakh Calcium Calcium sensor in astrocytes (GCaMP6f), membrane-tethered Khakh Lab pZac2.1...), diacylglycerol and STIM1 signalling system regulates directed cell migration. Nat Cell Biol. 2014 Feb...ER-targeted CEPIA1er ER-mitochondria tethering by PDZD8 regulates Ca(2+) dynamics in mammalian neurons. Science.... Mg(2+) Ions Released from ATP-Mg Hydrolysis Contributes to Mitotic Chromosome Condensation. Curr Biol...the adenosine sensor GRAB_Ado1.0 in neurons or astrocytes, (Constitutive or Cre-dependent) Li Lab GRAB_...

...including: how closely homologous the off-target sites are compared to the on-target site, the specific...immediately adjacent to the nearly-homologous target sites. The mere presence of additional PAM sequences should...selection tool that should be helpful in minimizing or testing off-target DSB. Is it possible to target a single...summarize HR template vector design guidelines? A few notes below are considerations for designing HR donor....with PX330 plasmid, clone in your target guides, test run them and then select the best guide to co-transfect...site in the donor. For example, choosing target sites that span the knock-in gene. For making mutations...As a successful insertion will destroy the BbsI sites, a double digest should discriminate between positive...

...function of zebrafish astrocytes. Knock-in tagging - Michel Bagnat lab. Targeting cassettes encoding fluorescent...Zebrafish Society (IZFS) - An organization that promotes and advocates for zebrafish research in the international...Morsch Lab. A vertebrate model to study ALS-linked aggregates in real time. Live imaging of astroctyes - Kelly...Resources Zebrafish Genome Project - Access the latest reference sequence assembly for the zebrafish genome...

...Institute Identifying Altered Epigenetic States and Drivers in Intestinal Carcinoid and Pancreatic Neuroendocrine... Cancer Institute Finding the Causes of Small Intestinal Neuroendocrine Tumors Ramesh Shivdasani Dana-...Dana-Farber Cancer Institute Epigenetic Regulators of Intestinal Endocrine Cells and Carcinoid Tumors Qiao Zhou...Zhou Harvard University Epigenetic Regulators of Intestinal Endocrine Cells and Carcinoid Tumors Bradley ...