...references are below. Pooled genome-wide human SAM library SAM can be combined with a human genome-wide library...please visit the Addgene SAM library page . Targeted gene activation using SAM There are two sets of 3 ...CRISPR Blog Posts Jump to: SpCas9 Plasmids GeCKO SAM AAV - In vivo Delivery AAV - Cas9 Mouse CRISPR ( ...information. Full references are below. Return to top Cas9 SAM transcriptional activation plasmids and screening...library CRISPR/Cas9 Synergistic Activation Mediator (SAM) is an engineered protein complex for the transcriptional...available for mammalian endogenous gene activation using SAM: Addgene plasmids #61422 - #61424 were designed for...sites for insertion of guide sequences Zhang Lab SAM Cloning Protocol 321.5 KB Return to top AAV - In vivo...

...70,290 SAM v1 - 3 plasmid system 1000000075 (Puromycin) Activation Mouse Zhang 3rd 3 69,716 SAM v2 - 2...3rd 1, 2, or 4 49,766 arrays Broad GPP kinome Brunello 75314, 75315 (1 plasmid) 75312, 75313 (2 plasmid... Library 141438 Knockout Human Yeo 3rd 10 12,472 SAM v1 - 3 plasmid system 1000000057 (Zeocin) 1000000074...GPP genome-wide Brunello 73179 (1 plasmid) 73178 (2 plasmid) Knockout Human Doench and Root 3rd 4 76,441...Broad GPP genome-wide Brie 73632 (1 plasmid) 73633 (2 plasmid) Knockout Mouse Doench and Root 3rd 4 78,637...172651 (Set D) Knockout Human Doench and Root 2nd 2 40,710 Inzolia Human CRISPR/Cas12a Multiplex Knockout...3,052 Broad GPP kinome Brie 75317 (1 plasmid) 75316 (2 plasmid) Knockout Mouse Doench and Root 3rd 4 2,852...

... has increased stability and light output Sanjiv Sam Gambhir 104587 pHIV-iRFP720-E2A-Luc Firefly EF1α ...fusing NanoLuc® and Venus fluorophore to Troponin C Ca 2+ binding domain. Nano-lanterns , Enhanced Nano-lanterns...transfection Feng Zhang 21471 pLenti PGK V5-LUC Neo (w623-2) Firefly PGK Lentiviral expression of firefly luciferase...

...interface upon thaw (Fig. 2). Before thawing, all the samples were at the control pH of 7.4 as indicated by ...showed that AAV infectivity was not altered by a short 2-hour exposure to pH as low as 2.5 (Potter et al., ...Methods. 2013 Apr;10(4):278-9. PubMed PMID: 23538862. 2. Potter M, Lins B, Mietzsch M, Heilbronn R, Van Vliet...affected the pH of our viral samples. Read on to learn how aqueous samples might be affected by dry ice...Measuring the pH of our samples: What we did To measure the pH of our samples, we used a solution of cell...when the samples were frozen. This was clear by the gradual change in pH visible at the sample interface...at -80°C. For samples that are shipped on dry ice, this can affect the pH of the sample, which may have...

...different activators exist, including the multi-plasmid SAM system. Select Expression System To use CRISPR, you...either (1) a lack of gRNA and/or Cas9 expression or (2) a lack of efficient target cleavage in cells expressing...efficacious than gRNAs containing a C nucleotide at the same position in spite of being a perfect match for the...

...domains used in SAM. This method has been shown to activate genes at 2- to 3-fold higher than SAM, SunTag, and...activation. Synergistic Activation Mediator (SAM) Description SAM uses specially engineered sgRNAs to increase...re-engineer components of Cas systems themselves. SunTag, SAM, and VPR have all shown significant improvements ... p65). Performance When targeting single genes, SAM consistently shows the highest levels of gene activation...regulation (activating multiple genes at once), however, SAM exhibits activation levels comparable to other popular...activators while showing lower activation levels than SAM. One drawback of this method is its construction:...VP64 activator, but lower levels compared to the SAM system. Its activation levels are similar to that...

...CRISPR/Cas9 Synergistic Activation Mediator or SAM. SAM is an engineered protein complex for the transcriptional...VP64 fusion; 2. A sgRNA incorporating two MS2 RNA aptamers at the tetraloop and stem-loop 2; 3. The MS2...not been achieved.” That is, until now. Enter the SAM pooled library In a paper published in Nature in ...plasmid which expresses the activation helper protein. SAM can be combined with a human genome-wide library ...Nature paper reports, “Here we have shown that the SAM system is robust, specific, and can facilitate genome-scale...combined with a compact pooled sgRNA library. Our SAM-mediated screens exhibited a high degree of consistency...References & Further Reading: For more information about SAM, including a tool for getting optimized guide sequences...

...Dilute oligos 1:10 in ddH 2 O ( e.g., 1.0 μl annealed oligos + 9.0 μl ddH 2 O to yield a concentration...been included. Ensure there are 2 x 10 6 cells per CRISPR pair. Resuspend 2 x 10 6 cells in 100 μl of electroporation...), 50-100 ng gDNA, and H 2 O up to 20 μl. Use the primers designed in step 2 above. Conduct PCR for “non-deletion...Run samples on 2% agarose gel at 10 V/cm using 1x Tris-acetate-EDTA (TAE) buffer. Examine samples for .... Pick 2 - 3 colonies and inoculate into a mini-prep culture. Perform mini-prep for each sample and sequence...deletion. Run sample in thermocycler and run the following program: 65 °C for 6 min, 98 °C for 2 min to extract... gDNA from clones. Run sample in thermocycler: 65 °C for 6 min and 98 °C for 2 min to extract gDNA. Screen...

...fusion none 2, 5 Richie 121539 pAAV SYN1 HA-hM3D(Gq) hM3D(Gq) - Activation HA fusion none 2, 5 Richie 50467...none 2, 8 Roth 50476 pAAV-CaMKIIa-hM3D(Gq)-mCherry hM3D(Gq) - Activation mCherry fusion none 1, 2, 5, ...(Gq) - Activation mCherry fusion Cre-dependent 1, 2, 5, 8, 9, rg*, PHPeB Roth 44362 pAAV-hSyn-DIO-hM4D...(Gi) - Inhibition mCherry fusion Cre-dependent 1, 2, 5, 8, 9, rg*, PHPeB Roth 65417 pAAV-hSyn-dF-HA-KORD-IRES-mCitrine...mCherry hM3D(Gq) - Activation mCherry fusion none 1, 2, 5, 8, 9, rg* Roth 50475 pAAV-hSyn-hM4D(Gi)-mCherry...mCherry hM4D(Gi) - Inhibition mCherry fusion none 1, 2, 5, 8, 9, rg* Roth 52536 rAAV-CAG::FLEX-rev:: hM4D-...mCherry hM4D(Gi) - Inhibition mCherry fusion none 1, 2, 5, 8, 9 Roth 75033 pAAV CD68-hM4D(Gi)-mCherry hM4D...

...of basal H 2 O 2 levels with peroxiredoxin-based probes Real-time monitoring of basal H 2 O 2 levels with...Belousov Hydrogen Peroxide (H 2 O 2 ) Cytosolic or mitochondrial sensor for H 2 O 2 oxidation (roGFP2-Orp1) ...peroxiredoxin-2-based probe. Nat Commun. 2018 Aug 7;9(1):3145. Hadley Sikes Hydrogen Peroxide (H 2 O 2 ) Monitoring...encoded Ca(2+) indicator with enhanced two-photon absorption. Neurophotonics. 2024 Apr;11(2):024207. doi... Red fluorescent calcium sensors (fRCaMP1/2, GECO1/2) The kinetic mechanisms of fast-decay red-fluorescent...intracellular Zn(2+) homeostasis. Nat Methods. 2009 Oct;6(10):737-40. Maarten Merkx Zinc eZinCh-2 Zn2+ FRET ... eZinCh-2: A Versatile, Genetically Encoded FRET Sensor for Cytosolic and Intraorganelle Zn(2+) Imaging...

...immunoglobulin heavy diversity 2-15 D2, IGHD215 IGHD2-2 immunoglobulin heavy diversity 2-2 IGHD22 IGHD2-21 immunoglobulin...heat shock 70kDa protein 2 HSP70-2, HSP70-3 HSPA4 heat shock 70kDa protein 4 APG-2, HS24/P52, MGC131852, ...variable 2-33 (non-functional) IGLV233, V1-9 IGLV2-8 immunoglobulin lambda variable 2-8 IGLV28, V1-2 IGLV3...beta 4 DEFB-2, DEFB102, DEFB2, HBD-2, SAP1 EDN1 endothelin 1 ET1, HDLCQ7 EDN2 endothelin 2 ET2, PPET2 ...receptor subfamily 2, group C, member 1 TR2 NR2C2 nuclear receptor subfamily 2, group C, member 2 TAK1, TR2R1...suppression of tumorigenicity 2 - STC1 stanniocalcin 1 STC STC2 stanniocalcin 2 STC-2, STCRP TAC1 tachykinin,...FLJ99988, FLT2, HBGFR, KAL2, N-SAM, OGD FGFR2 fibroblast growth factor receptor 2 BEK, BFR-1, CD332, CEK3, ...

...extraction protocol for more details.   Figure 2: An uncut plasmid is expected to form 3 bands: nicked...replicate.   How can I prevent recombination in my samples? Plasmid recombination can affect viral vectors...stable E. coli reduce the rate of recombination in samples. For a more detailed comparison of these cloning... easiest and clearest ways to determine if your sample has recombined. Choose a restriction enzyme that...

...111707 Mode #2 Library (NEDD4 WW2 domain) Pooled Library 111708 Mode #2 Library (NEDD4-2 WW2 domain) References...Pooled Library 111705 Mode #2 Library (14-3-3β) Pooled Library 111706 Mode #2 Library (14-3-3σ) Pooled Library...phosphorylation-dependent protein-peptide interactions (“mode #2” expression, Barber et al, Nat. Biotech. 2018). The...fluorescence-activated cell sorting (FACS). The mode #2 phosphosite library with four different phosphobinding... WW2 domain of NEDD4, and the WW2 domain of NEDD4-2; 111705-8 ) is available from Addgene. Screening kinases...iSPI_pSer_Subpool#1 Pooled Library 188527 iSPI_pSer_Subpool#2 Pooled Library 188528 iSPI_pSer_Subpool#3 Pooled Library...pos ctrl Plasmid 111883 pNAS1b split mCherry NEDD4-2 WW2 Plasmid 111882 pNAS1b split mCherry NEDD4 WW2 ...

...that numbers are not switched around or misread.  2. Track the container - It is just as important to ...Every plasmid sample enters Addgene the same way. A package is delivered by a mail courier and then the.... Some samples are submitted as bacterial colonies in petri dishes, but close to 80% of samples are received...every sample can be tracked easily at each step in the storage process. After the original sample is barcoded...scanned to verify that it is the correct sample. Tracking samples - it's all about location, location, location...these samples have the 2D barcodes mentioned above. These barcodes identify what type of sample is contained... on the sample type. It is critical for us to know which plate, rack, and freezer each sample is located...

...suggests that titers obtained using ddPCR are generally 2–3 fold higher than those achieved using the standard...catalog item (see maps section for images). Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre... a new vial of early passage cells. Approximately 2 weeks post-thaw, cell culture supernatant is tested...of the viral capsid proteins VP1, VP2 and VP3. Samples of viral preparations are subjected to polyacrylamide...proteins as a fraction of total protein present in the sample is also determined and used to determine purity...shown relative to the total protein present in the sample. Sterility Viral vector preparations are added ...rigorous barcode matching system that follows the sample from the DNA tube all the way to the cleared viral...

... mutations in the gene methyl-CpG binding protein 2 ( MECP2 ). MECP2 Rett syndrome is an X-linked disorder...X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet . 23, 185–188. (Link opens in a new window... Cuddapah et al. 2014. Methyl-CpG-binding protein 2 (MECP2) mutation type is associated with disease severity... Specific mutations in methyl-CpG-binding protein 2 confer different severity in Rett syndrome. Neurology...males with mutations in Methyl-CpG binding protein 2. Am J Med Genet B Neuropsychiatr Genet . 180, 55–67...variability in symptoms even within patients with the same mutation, the location and type of MECP2 mutation...cell line repository (Link opens in a new window) Sampled - one of the world's largest biorepositories (Link...

...strategies for CRISPR-HIV therapeutics - one using dCas9-SAM to activate HIV-1 transcription and destroy infected...realistic with the advent of CRISPR. Using CRISPR/Cas9 SAM to "shock and kill" To “shock and kill” the HIV-...the CRISPR/Cas9 Synergistic Activation Mediators (SAM) system available from Addgene. This system uses ...genome, which acts as a promoter, and found that SAM targeting of the enhancer region near the NF-KB binding...latent microglial line, treatment with CRISPR/Cas9 SAM increased the percentage of cells with activated ...that can produce HIV-1 toxic proteins, CRISPR/Cas9 SAM caused apoptosis, indicating a true “shock and kill...CRISPR/gRNA-directed synergistic activation mediator (SAM) induces specific, persistent and robust reactivation...

...overview of the most common methods. BioBrick BioBrick-2 BglBrick Silver Standard Freiburg Standard BioBrick...visit iGEM: BioBrick (RFC 10) Back to Top BioBrick BB-2 (RFC 12) Prefix: GAATTC GCGGCCGC T ACTAGT G Prefix...XbaI, SapI For more info, visit iGEM: BioBrick BB-2 (RFC 12) Back to Top BglBrick – Berkeley Standard ...assembly, even as more parts are added. Therefore, the same reaction which ligates a promoter into an empty ...

...sites on the same chromosome. Most multiplex systems enable researchers to target anywhere from 2–7 genetic...protein, instead of a two-component system (Figure 9C) SAM activators - co-expression of dCas9-VP64 with a modified...left free to interact with the target DNA. Figure 2: Overview of the NHEJ repair mechanism Cas9 will only...Cas9 is included in the gRNA-containing plasmid, or 2-vector systems, in which Cas9 must be delivered separately...your experimental cell population (Figure 8E). In a 2-vector system, you’ll need to either co-infect with...the presence of infectious organisms (like SARS-CoV-2 ) and genetic mutations. Similar to Cas9 and Cas12...Cas enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme...

...cells; function is unknown Monomer with a valency of 2 IgE Protects against parasites and is responsible ...such as anaphylactic shock Monomer with a valency of 2 IgG The most abundant isotype in blood, also found...secondary immune response Monomer with a valency of 2 IgG1; IgG2; IgG3; IgG4 IgM Produced during the primary... ranging from antibody fragments (shown in Figure 2) to completely unrelated molecules. Examples include...used for assembling protein nanostructures. Figure 2: An antibody (IgG, far left) compared to alternative...application Collect and lyse samples to make proteins available. Tissue samples may need additional processing...culture samples to extract proteins. Homogenize (tissues only) and lyse. Cross-link the samples with formaldehyde...