...Ken-Ichi Takemaru 122871 GFP-hCCDC11 Centriolar satellites CCDC11 EGFP Ken-Ichi Takemaru 54234 mEmerald-...

...your final RNA sample(s) are resuspended in water or TE buffer, store them in a -80°C freezer to prevent ...

...readings than a DNA sample dissolved in buffer (such as TE). You will get much more accurate and consistent ...ranges from 1.80-2.00). Repeat for each sample. Notes: Keep in mind that despite the accuracy of the NanoDrop...

...Ousterout DG, Leong KW, Guilak F, Crawford GE, Reddy TE, Gersbach CA (2013) RNA-guided gene activation by...

...have been used to visualize “thorny excrescence” (TE) spines in the Hippocampal CA3 pyramidal neurons ... The central nervous system (CNS) orchestrates complex processes enabling organisms to control their ... affects the labeling of small structures like neurites. Like epitope tags, low FP expression may be insufficient...structures through their multiple, high-affinity binding sites for primary antibodies which can be used to amplify...difficult to label. It has also been shown that dendrites in the stratum oriens and the stratum lacunosum...their multiple, highly specific antibody binding sites and primary antibody-based detection, smFPs create...

...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 ...

.... https://doi.org/10.1038/s41592-019-0614-5 Sztal TE, Stainier DYR (2020) Transcriptional adaptation: ...inverted repeats separated by a loop region that facilitates the formation of a hairpin RNA structure, resulting... gene of interest. Tools for RNAi Arguably the greatest advantage of RNAi is the straightforward nature...active development, the first step necessarily dictates the direction and scope of the subsequent two....Artificial micro RNA and short hairpin RNA The greatest limitations of the long hpRNA approach are thought...experiments typically involved antibiotic selection to test for the presence of the RNAi transgene, followed...Savitski M, Grandi P, Jakob P, Michon A-M, Sun H, Tessmer K, Bürckstümmer T, Bantscheff M, Steinmetz LM, ...

...victoria Chimera: Mouse/Rat rat IgG2a Addgene distributes ready-to-use recombinant antibodies made from...

... PRKN mTurquoise2 CMV Parkinson's Pablo Rivera-Fuentes 111723 pBacMam2-DiEx-LIC-N-flag_huntingtin_full-length_Q23...

...enzyme sites. You can easily move (subclone) any piece of DNA that already has restriction sites on either...systems is that like restriction enzyme sites, type IIS sites can be found throughout DNA sequences. Thus...double stranded DNA (dsDNA) at specific sequence sites in the DNA. In restriction cloning, scientists utilize...either side of it into any plasmid that has the same sites in the same orientation within its multiple cloning...short length, it is also easy to add restriction sites to any piece of DNA during PCR amplification, allowing...important to note that restriction enzyme target sites can be repeated throughout a specific DNA sequence...already be surrounded by specific recombination sites (in this regard, not so dissimilar from restriction...

...of DNA, or templates, are spatially separated from each other. These attached DNA templates are then amplified...Alternative start codons include CUG in eukaryotes and GUG in prokaryotes. Common Epitope Tags Tag Amino Acid...DNA. Although they can be found in archaea and eukaryotes, they play the most significant biological role...fragment with a plasmid or vector DNA backbone generates a recombinant DNA molecule, which can be used ...of DNA which contains several restriction enzyme sites, enabling easy insertion of DNA by restriction enzymes...the promoter. As a general rule, the restriction sites in the MCS are unique and not located elsewhere ...relatively quickly, and exhibit exponential growth rates, plasmids can be replicated easily and efficiently...

... transcriptional units and facilitates integration. LEDGF/p75 binding sites are enriched in gene bodies...shuffling restriction sites between vectors until the gene of interest is flanked by sites compatible with ... that contain the restriction sites needed. Functional restriction sites must be a certain number of bases... always SIN: Uses 4 plasmids instead of 3 and eliminates the requirement for Tat. LTR Viral Promoter Wild...cells are usually used to produce lentivirus. What dictates lentiviral host cell range (tropism)? Lentiviral...target species. Although chromatin availability facilitates integration, it does not explain the lentiviral... subcloning or appending compatible restriction sites onto the insert of interest using PCR. The process...

...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...

...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...

...the other hand, the process in eukaryotes is much more complex. Eukaryotes require a minimum of seven transcription...actually begins. Only eukaryotes and archaea, however, contain this TATA box. Most prokaryotes contain a sequence...are common in prokayotes, specifically bacteria, but have also been discovered in eukaryotes. Operons consist...binding is very different in bacteria compared to eukaryotes. In bacteria, the core RNA polymerase requires...Transcription is tightly controlled in both bacteria and eukaryotes. Promoters are controlled by various DNA regulatory...promoter also contains transcription factor binding sites, but mostly contains regulatory elements. Eukaryotic...Specific Drosophila promoter containing Gal4 binding sites Bacterial Promoters Promoters in bacteria contain...

...attB sites, the phage integrates into the bacterial genome flanked by two new recombination sites (attL-left...reactions. Recombination of attP and attB sites creates attL and attR sites.   Gateway vectors contain modified...takes place between the attB sites flanking the insert and the attP sites of the donor vector. This reaction...enzyme mix and generates the entry clone containing the DNA of interest flanked by attL sites. As a byproduct...reactions. The BP reaction creates an attL-flanked entry clone. The LR reaction creates an expression clone ... place between the attL sites of the generated entry clone and the attR sites of the destination vector...attB, P, L and R sites that recombine very specifically and directionally: attB1 sites react only with ...

...and astrocytes Mice No (LY6C1)   Hanlon et al., 2019 AAV-BI-28 CNS: neurons, astrocytes, oligodendrocytes.... It can also effectively transduce astrocytes or oligodendrocytes when paired with appropriate cell type-specific...28 has been tested in C57BL/6J mice and effectively transduces NeuN+ neurons and astrocytes (Huang et al...et al., 2023). Among the capsids tested, AAV9-X1.1 showed the greatest fold change in RNA and DNA levels... others have done, you’ll need to experimentally test your chosen capsid before using it in an application...elements [promoters and enhancers, miRNA target sites, and recombination target sequences, denoted by ...cisterna magna or lumbar spine. Image and caption courtesy of Challis et al., 2022. Where do they come...

... Cre but lox511 sites can only recombine with other lox511 sites, not with loxP sites. Alternatively, ...at target sites to induce site specific recombination events: Cre recombinase binds loxP sites, while FLP... FLP binds FRT sites. When a DNA sequence is flanked by target sites (floxed) in opposing orientations..., you could use a pair of loxP sites and a pair of FRT sites and include both Cre and FLP recombinases...proceed either by first utilizing the loxP sites or the lox551 sites. Regardless, the first recombination step...fragment using either loxP or lox511 sites, leaving two identical sites on one end of the DNA fragment (Figure..., and that the orientation of the flanking loxP sites directs the Cre recombinase to invert, translocate...

...recombination one of lox2272 sites will now be sandwiched between 2 loxp sites and is excised by Cre (Figure...orientation and one of the unused (loxp) sites will be flanked by two lox sites now in the same orientation. Next...recombination sites, they hypothesized that spontaneous recombination between these sites may be the cause...leaky expression. To test their hypothesis, they randomly scrambled one pair of lox sites and either used ...injection site. Test the promoter It is also essential that the promoter be thoroughly tested in your cells...fibrillary acidic protein (GFAP) for astrocytes it is important to test it in the brain region of interest...transgene is floxed by two pairs of recombination sites and the open reading frame is inverted in relation...

...do some testing or ask experienced colleagues.   Figure 1: The laser pulse illuminates the cell... the next gates fully depend on your individual experiment. Practical example: lymphocytes in a mouse...first three gates out of the way, it’s time to move on to my experimentally-specific gates. Two-dimensional...free to use multiple individual gates. Note that in this case, square gates were used.  Congratulations...distinguish single cells from duplicates. Lastly, you learned which gates should always be included in ...samples, it is necessary to set up a sequence of gates to be able to select and precisely measure your ...in cell size, marker expression, and viability. Gates are the parameters the machine uses to differentiate...