...from 40 mM to 100 mM NaCl but stained evenly in 1x TE buffer. The lab also used tTALE-FP in a single molecule-fluorescence...infection, because this setup allows for experimental replicates for each of the gRNAs. The idea was to assign...The SPARC system uses competitive recombination sites to selectively remove a stop cassette upstream of...expressed. By truncating one of the recombination sites in the construct to different lengths, the density... of Ca2+ are high in the cell, calcineurin dephosphorylates the protein CZI, which in turn binds to calcineurin-dependent...

... AAV-PR encodes AAV-PR capsid that transduces pericytes and smooth muscle cells in mice after systemic...

...-Beltran WF, Lam EC, Astudillo MG, Yang D, Miller TE, Feldman J, Hauser BM, Caradonna TM, Clayton KL, ...neutralized. The B.1.617.2 (Delta variant) was not tested at the time of this study. Figure 1: Neutralization...cross-neutralization occurs in the P.1 and B.1.351 variants tested. Image from Garcia-Beltran et al., 2021 with permission...happening.”  In their assay, the neutralization rates of B.1.351 pseudovirus was similar to that of pseudoviruses...

...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 ...

...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...

...Anti-VAChT [N425/45R-1] VAChT Rat Mouse IgG1 Addgene distributes ready-to-use recombinant antibodies made from...

.... 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, ...

... bioassay plates and remove bacteria. Use one scraper for all plates. Use two 10 mL pipettes (one for ...Antibiotic 245 mm bioassay plates (Molecular Devices, X602) *Pro-Tip* Pour these plates at least one day in ...Agar + Antibiotic plates. Prewarm 12 mL recovery media at 37 °C (for at least 15 minutes). Prewarm 3X LB... Agar + Antibiotic plates at 37 °C. Prewarm 8X LB Agar + Antibiotic Bioassay plates. Prechill Micropulser...Micropulser cuvettes on ice. Thaw 4 tubes of electrocompetent cells on ice for 15-20 minutes or until completely...transformed cells on each of the eight bioassay plates (two plates per tube). Distribute evenly with a sterile... edge that can scrape plates more abrasively at a certain angle. Incubate plates upside down at 30 ℃ overnight...

...existing sites in the original vector. Designing oligos: To add NdeI, PacI, AscI and MfeI sites between...EcoRI and SalI sites of the vector, we design a top oligo with each of the additional sites in tandem ( ... to start at 95°C for 2 minutes. Then, gradually cool to 25°C over 45 minutes. Ligation: Dilute 5μL of...tutorial we will discuss how to add new restriction sites to the MCS of an empty vector. However, the following...set of oligos containing your desired restriction sites and add them to your existing vector. It is a couple...oligo), but this would destroy the EcoRI and SalI sites in the final vector. Order the following oligos ...Place tube in 90-95°C hot block and leave for 3-5 minutes. Remove the hot block from the heat source (turn...

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

...dilution is appropriate). Quality of duplicates: Exclude duplicates from analyses if there is more than... 2 hours qPCR run: 1.5 hours Data analysis: 30 minutes Equipment qPCR instrument Heating plate Pipettors...water Microcentrifuge tubes 96-well optical plate Pipettes Pipette tips General Considerations Always run...-log of the expected titer of the samples being tested. Always include a No Template Control (NTC), i....small aliquot of each standard (enough for 1 or 2 plates) and store at -20°C. Once a standard is thawed ...the assay multiple plasmids containing ITR were tested. Plasmid #59462 is one plasmid that gave reliable...consistent results. Use the recommended plasmid, or test multiple plasmids to find a suitable one. Some labs...

...be worn at all times. Use plastic pipettes in place of glass pipettes or needles. Liquid waste should be...shRNA oligos are cloned into the AgeI and EcoRI sites in place of the stuffer. The AgeI site is destroyed... 10x NEB buffer 2 35 μL ddH 2 O Incubate for 4 minutes at 95°C in a PCR machine or in a beaker of boiling...PCR machine, incubate the sample at 70°C for 10 minutes then slowly cool to room temperature over the period...following manufacturer’s protocol. Plate on LB agar plates containing 100 μg/mL ampicillin or carbenicillin...swirling or gently flicking the tube. Incubate for 5 minutes at room temperature. e. Add 80 μL of FuGENE® master...gently flicking the tube. f. Incubate for 20-30 minutes at room temperature. g. Retrieve HEK-293T cells...

...DNA for 2 minutes at 72°C. Repeat steps 8-10 for 25-30 cycles. Final Extension for 5 minutes at 72°C. ...oligo primers. This molecular biology technique creates several micrograms of target DNA from just a few.... Basic PCR Program Initial Denaturation for 2 minutes at 94°C: This initiation step heats the double ...Repeat steps 2-4 25-30 times. Final Extension for 5 minutes at 72°C: A final extension to fill-in any protruding...melting temperature (Tm). Set extension step at 1-2 minutes per kilobase of product depending on whether you...time and temperatures. Initial Denaturation for 2 minutes at 94°C. Denature for 30 seconds at 94°C. Anneal...denaturation of the DNA, particularly for GC-rich templates. What does each ingredient specifically do? Template...

...restriction sites to flank your insert : You can use PCR Based Cloning and add restriction sites to the ends...as long as it is already bounded by restriction sites that are also present in the same orientation...Analyzer , allow you to identify which restriction sites are present in a given sequence. When selecting ...recipient plasmid as well as a specifically designed test digest later to verify that the insert was cloned...a version of your insert flanked by restriction sites compatible with the recipient plasmid's MCS. However... within your insert. Adding desired restriction sites to your recipient plasmid : You can modify the MCS...

... fits the plate. Incubate plates at 37 ℃ for 18 hours. Day 2 Observe plates for colony formation. Shown...Antibiotic Plating You may also like... Making LB Agar Plates Bacterial Transformation Recovering Plasmid DNA...source) Incubator Reagents 6 cm diameter LB/agar plates without antibiotic High concentration (100 mg/mL... the plate at room temperature for at least 30 minutes with the lid on to give the antibiotic time to ... with smaller size than the 1 mg/mL and 2 mg/mL plates and effective selection. Selection Curve of Transformed...

...Making LB Agar Plates Create plates to culture bacteria in the lab Pouring LB Agar Plates Protocol Streaking...Bacteria on Plates Isolate single bacterial colonies on an agar plate Streaking Bacteria on Plates Protocol...CRISPR Protocol Over-Agar Antibiotic Plating Create plates to culture bacteria in the lab Over-Agar Antibiotic...

...video for tips on pipetting in the lab. Equipment Pipettes Pipette tips Waste container Containers to hold...Tips Although there are many different types of pipettes that have different mechanisms to disperse liquids...liquids, this protocol is focused on single channel pipettes. These come in standard sizes: P2, P10, P20, P200...would read as 050. Each volume display on these pipettes will also have small tick marks at the bottom....Liquids of various viscosity have different flow rates. The more viscous a liquid, the lower the flow rate...’s important for scientists to calibrate their pipettes to make sure that they are properly tuned to dispense...

..., VWR 76322-154 Pipettes, 5 mL, VWR 89130-896 Pipettes, 10 mL, VWR 89130-898 Pipettes, 25 mL, VWR 89130...of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment...89130-900 Pipettes, 50 mL, VWR 89130-902 Gel loading tips, Corning 4853 Microcentrifuge tubes, Neptune ...mobility of the bands (sample AR0016 in Figure 1) indicates that the samples may not have been processed correctly...