... that results in a polymerase error rate of up to 2% per nucleotide position. The “sloppy” PCR reaction...modifications: 1) increased concentration of Taq polymerase, 2) increased PCR extension time, 3) increased concentration...to synthesize a complementary mutant strand (step 2 and 3). Then the uracil-labeled template strand is...restrict site matter? Most of the time no, but there are 2 instances where it does. 1) When the backbone vector...ExoIII. See the below figure for an example like this. 2) When your gene of interest has a BbvCI site(s). If...saturation mutagenesis method are 1) ease of use, 2) time savings, and 3) ability to perform single site...PMID: 23766324. PubMed Central PMCID: PMC4409680. 2. Mccullum, E. O., Williams, B. A., Zhang, J., & Chaput...

...the mixed sample in our pipeline (see Figure 2, sample 2). When we blindly analyzed AAV-68544, we were...look for potential contaminants. The analysis is a 2-step process using Geneious software: First, the individual.... When contamination is suspected, we proceed to de novo assembly of the unmapped reads, employing our...transduced cells whenever possible. This post will describe our workflow for viral genome sequencing (VGS)...

.... Okita et al describes a protocol for generating iPSCs by plasmid transfection where 2 of 11 clones tested... cell reprogramming factors and wait for cells to de-differentiate. However it’s difficult, especially...plasmid-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming factors of...understanding the mechanisms of reprogramming, and 2) studies with clinical endpoints. In the first scenario...PubMed PMID: 27764162. PubMed Central PMCID: 5072705. 2. Sommer, Cesar A., and Gustavo Mostoslavsky. "The ...challenges." Journal of Cellular Physiology 228, no. 2 (2012): 267-75. PubMed PMID: 22767332.  3. Kim, Dohoon...PubMed PMID: 23546745. PubMed Central PMCID: 4176696. 2. Yusa, Kosuke, Roland Rad, Junji Takeda, and Allan...

... be extracted from small bacterial cultures (i.e. 2-3 ml) with or without commercially available kits ...ratios measured at 260 and 280 nm should fall ~1.8 - 2 and give scientists an indication of purity versus...contaminants. The 260 and 230 nm ratios should fall ~2 - 2.2, and provide an indication of purity versus ...hydrochloride used during plasmid extraction. Figure 2: Use cloning grade DNA for direct cloning of your ...other uses.   Cloning grade DNA Molecular cloning includes a set of techniques that are used to insert recombinant...

...non-homologous end joining (NHEJ) (2-5), depending on your desired genome edit. Since exploitation of ...each sample can be quickly calculated (details in [2]) and can be used to quantify rates of mutation in... have been reported for mismatch nuclease assays (2, 6, 7). Ability to distinguish between mono-allelic...to testing their performance (2). Remember that although digital PCR is a great screening tool, it still...PMID: 24509632. PubMed Central PMCID: PMC4063274. 2. Findlay, S. D., Vincent, K. M., Berman, J. R. & Postovit...your genome edits, this paper provides detailed instructions on how to design such assays, in addition to...great deal of precision. This means digital PCR provides an absolute measure of target abundance, does ...

...systems: a burst of class 2 and derived variants. Nature Reviews Microbiology, 18(2), 67–83. https://doi.org...online, searchable resource that provides summary information on Class 2 Cas enzymes. The information is...this content and much, much more.     Fig. 2 – List of resources and properties for a Cas entry...Database: A Functional Classification System for Class 2 CRISPR-Cas Enzymes [Preprint]. Biochemistry, Biophysics... is also documented. Experimental design When you are in the design phase of your experimental planning...is organized in wiki format and includes biochemical properties, previously established uses, and intended...relevant resources The homepage displays a graphic describing CasIDs and relevant properties of Cas enzymes...

...Table 2 for more details on the effect of pH on the fluorescent properties of Rosella.   Table 2: Effect...in yeast. Autophagy,4(2), 205-213. doi:10.4161/auto.5331. PubMed PMID: 18094608 2. Sargsyan, A., Cai, J...environment like the vacuole or lysosome. See Table 2 for a summary of what color fluorescence Rosella emits...fluorescence accumulates in the vacuole. See Figure 2 for an example of what fluorescence looks like for... phosphatase activity: In yeast, the PHO8 gene encodes a vacuolar alkaline phosphatase. Normally PHO8 ...

...them in Regenerative Medicine. Acta Naturae, 2010. 2(2): p. 18-28. PubMed PMID: 22649638. PubMed Central...capability to differentiate into specialized cell types [2]. We general speak of 4 levels of stem cell potency...zygote through to the morula stage are totipotent [2-4]. Pluripotency: When a cell has the ability to differentiate...stem cells (ESCs), which I discuss in detail below [2]. Multipotency: When a cell can differentiate into...PMID: 26726264. PubMed Central PMCID: PMC4686367. 2. Jaenisch, R. and R. Young, Stem cells, the molecular...essence, pluripotent cells can be thought of as descendants of totipotent cells. An example of pluripotent... produced Ethical concern High. Embryos are destroyed during ESC isolation. Low Chances of post-transplantation...

...and Amy tell us a little about themselves. Chapter 2: What is Science in the News (SITN)? - SITN is a graduate... discusses some of the programs SITN runs and describes some of the more popular topics from the Science... fields as well as students from the ed school, design school, and Kennedy school to discuss the impacts...impacts of science in a social context. Episodes are driven by narrative and story telling. Chapter 5: The...The SITN Blog - Vini and Amy describe how the SITN blog is a source of scientific information for the ...underwater? Chapter 8: Work-Life Balance - Vini describes how she balances working in the lab and working...

...different destination vectors depending on the total number of gRNAs you wish to clone, from 2-7. For example... plasmids allow expression of 2-5 gRNAs in zebrafish. Custom destination vectors are used depending upon...illustrated in the schematic below.   Figure 2: gRNA target sequences (colored rectangles) are cloned... plasmids: This plasmid set allows you to express 2-4 gRNAs, with four being the ideal number. First you...gRNAs into spectinomycin-resistant plasmids pX330S-2 to pX330S-(last gRNA number). The 5’ most gRNA is ...Multiplexing Plasmids: These plasmids allow you to assemble 2-4 gRNAs through Golden Gate or Gibson Assembly. gRNAs...and a subsequent curing protocol that requires only 2-3 hours incubation. Kondo Lab multiplexed base editing...

... H. J. (2009). Rats! Disease Models & Mechanisms, 2(5–6), 206–210. https://doi.org/10.1242/dmm.002733 ...pathological developments (Vieira et al., 2019) Besides their ability to regenerate, the axolotl has several...scientific history, highly characterized genome, and widespread utilization, Arabidopsis has made significant...Arabidopsis genome project marked the beginning of widespread Arabidopsis genetics research development. Arabidopsis...allows for easy cloning of constructs specifically designed for use in Arabidopsis. There are also important...

...Manitoba due to their Biosafety Level-2 or 2+. Please Note: Despite the fact that AAVs themselves are not...long and stable expression (up to 9 months in mice [2]). Low Immune Response: Compared to other viral vectors... location. Location (1): What tissue? Location (2): Where in the tissue/which cell type? Location (3...may be sufficient to overcome the transport issues. 2. Failure to plan (for storage) Is planning to fail...Find Ready-to-Use Retrograde AAV Preps at Addgene 2) Comparison of genetically engineered receptors (i.e...Therapy 15.10 (2007): 1740-1743. PubMed PMID: 17882145. 2. Zincarelli, Carmela, et al. "Analysis of AAV serotypes... tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain." PloS one 8.9 (...

...and materials required for each [1, 2]. This protocol [3] provides, in exquisite detail, the various steps...stimulation         In the video above, Channelrhodopsin 2 (Chr2) has been expressed in the motor cortex of a...lasers are relatively cheap and will run you about $2-3,000/laser. However, an important caveat is that ... the light-gated cation channel, Channelrhodopsin 2 (ChR2), exists in several states: closed/light receptive...PMID: 21628464; PubMed Central PMCID: PMC3138296. 2. Sparta DR, et al. Construction of implantable optical...board, Jacqueline N Crawley  [et al]. 2012;Chapter 2:Unit2 16. doi: 10.1002/0471142301.ns0216s58. PubMed...

...TUs. Level 2: Assembly of multiple Level 1 TUs into multigene constructs. Figure 2: The three ...can be queried by testing any number in the Level 2 assembly. These pathways can require a lot of optimization... of multigene constructs. PLoS One. 2011 Feb 18;6(2):e16765. doi: 10.1371/journal.pone.0016765.  Hahn,... (2020). https://doi.org/10.1186/s12870-020-02388-2 Resources on Addgene.org Addgene’s Modular Cloning...cloning, or MoClo, is a systematic cloning strategy designed by synthetic biologists to perform large scale...specifically refers to the organizational schema described above, and not any particular cloning technique...example of an individual part, this category also includes promoters, UTRs, terminators, etc. Once selected...

...revert back to a pluripotent stage (iPSC formation) [2, 3]. The iPSCs then proliferate and redifferentiate... more common delivery methods include retrovirus [2], lentivirus [4], adenovirus [5], Sendai virus [6]...2006. 441(7097): p. 1061-7. PubMed PMID: 16810240. 2. Takahashi, K., et al., Induction of pluripotent stem... Pluripotent Stem Cells. Cell Stem Cell, 2015. 17(2): p. 178-94. PubMed PMID: 26189426. 15. Si-Tayeb, ...induced pluripotent stem cells. Stem Cell Rev, 2015. 11(2): p. 242-53. PubMed PMID: 25578634. PubMed Central... to regeneration. Nat Rev Mol Cell Biol, 2011. 12(2): p. 79-89. PubMed PMID: 21252997. 37. Merrell, A.... and B.Z. Stanger, Adult cell plasticity in vivo: de-differentiation and transdifferentiation are back...

...fluorescent state make them useful for 2-color imaging with green PA-FPs. Table 2: Properties of Selected Reversible...tetroxide). These conditions destroy most PA-FPs, but, in 2015, the Looger Lab designed 2 variants of EosFP that... conventional FP imaging. PA-FPs are divided into 2 main categories - those with irreversible and reversible... forms of the chromophore which contribute to the 2 peaks of wtGFP’s excitation spectrum- a major 397 ... / N99K / S148L / I165V / Q167P / L169V / I203R) (2) and PA-mRFP1(S146H / I161V / I197H) (3) were derived...with two distinct wavelengths of light (see Table 2). The best known reversible PA-FP is the naturally... above background. The majority of PA-FPs come in 2 flavours- monomeric and tetrameric. Tetrameric PA-...

...pseudotransduction” — to describe the direct protein transfer of RT, which would actually occur in step 2 during uncoating...that have been developed into retroviral vectors and 2.) a subclass of retroviruses called the lentiviruses...effectively. Retroviral structure   Figure 2: Structure of a retrovirus. Image re-used from Dong...Dong & Kantor, 2021 under CC-BY license. Figure 2 gives a detailed view of the structure of a retroviral...one of the two strands’ DNA counterparts from step 2 will be integrated by the viral integrase protein ... Air. Environmental Science & Technology Letters, 2(4), 84. https://doi.org/10.1021/ACS.ESTLETT.5B00050...Biology (gDNA → mRNA → protein): their roadmap includes RNA → DNA (Cooper & Temin, 1974), as one of its...

... can be used to diagnose the presence of SARS-CoV-2 RNA, but inadequate access to reagents and equipment...named this system SHERLOCKv2 for SHERLOCK version 2. Here are some of the improvements: SHERLOCKv2 uses... a protocol for using SHERLOCK to detect SARS-CoV-2 RNA. The test is started using RNA purified from patient...recently adapted the DETECTR platform to detect SARS-CoV-2 using a lateral flow strip format. They published ...CY (2020) CRISPR–Cas12-based detection of SARS-CoV-2. Nature Biotechnology. https://doi.org/10.1038/s41587... and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems. Molecular Cell 60:385–397 . https...Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759–771 . https://doi....

... needing to work with a transfectable cell line. 2) They require larger expression vectors which are ...are also larger than their Cpf1 counterparts. Table 2. Cas9 Multiplexing Options  Multiplexing Method... Cpf1 expression vector.  See the graph in figure 2 for a comparison of editing frequency resulting from...Biotechnology 35.1 (2016): 31-34. Pubmed PMID: 27918548. 2. Zetsche, B., Gootenberg, J., Abudayyeh, O., Slaymaker...Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell,163(3), 759-771. PubMed PMID...editing!  A recent paper from Feng Zhang's lab describes how to use Cpf1 for multiplex genome editing.  ...sequence NGG TTTV PAM site preserved? Usually Destroyed Yes, Cpf1 cleaves 5' of the protospacer Multiplexing...

...improving editing accuracy by preventing re-editing and (2) optimizing “cut-to-mutation distance”. Furthermore...also takes into account that HDR is rare, usually 2-5% of the edited cells in our hands, these combined...screen a few hundred clones to find the right one Step 2 – Edit the cells a second time using a repair template...Additional CRISPR Resources on Addgene's CRISPR Guide Page 2. Optimizing "cut-to-mutation distance"  Even though... wisely (and let them cut close).   Figure 2: Relationship between cut site to mutation distance...PMID: 23287718. PubMed Central PMCID: PMC3795411. 2. Mali, Prashant, et al. "RNA-guided human genome engineering...overall HDR rates, we have shown recently that desired genome-editing events by HDR can be generated more...