...sequences from step B.1 into the oligos below. Do not change the ends; these bases are important for cloning...light. Short wavelength UV light will increase the chance of damaging the DNA. Purify the DNA using a Qiaquick...CO 2 for 12-15 hours. Day 3: j. In the morning, change the media to remove the transfection reagent. Replace...to cells. Days 3+: e. Examine cells each day and change to fresh puromycin-containing media every other...Target cells should be approximately 70% confluent. Change to fresh culture media containing 8 μg/mL polybrene...Incubate cells at 37°C, 5% CO 2 overnight. Day 3: e. Change to fresh media 24 hours after infection. TIP: If...control for the puromycin selection. Days 4+: g. Change to fresh puromycin-containing media as needed every...

...0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000...1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container pH ... one suitable with your protocols. FBS can be purchased already heat inactivated or it can be inactivated...panels: bright field images; right panels: GFP channel images. Last reviewed on: August 2, 2023...

...0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000...1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container Inverted... one suitable with your protocols. FBS can be purchased already heat-inactivated or it can be inactivated...

...0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000...1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container Reagents... one suitable with your protocols. FBS can be purchased already head inactivated or it can be inactivated...

...of CrChR2 590 Channelrhodopsins: cation channels from other species VChR1 Channelrhodopsin from Volvox ... mutations 540 Channelrhodopsins: chloride channels iChloC Chloride-conducting channel, CrChR2 with mutations...optogenetics plasmids for your experiments. Channelrhodopsins Channelrhodopsins are a foundational optogenetics tool... direct stimulation of ion channels. Naturally occurring channelrhodopsins were discovered in the green...chloride channels, these variants result in the hyperpolarization of neurons. Examples of anion channel variants...Jaws Browse Halorhodopsin plasmids . Archaerhodopsins Archaerhodopsin-3 (Arch) from Halorubrum sodomense...Description Peak Response Spectra (nm) Channelrhodopsins: cation channels from Chlamydomonas reinhardtii ChR2...

...0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000...1000 µL single channel pipette CO 2 incubator Pipet controller Hazardous waste container Microscope Hemocytometer... In the absence of selective medium, there is a chance that the integrated transgene could be lost through...

.... You can therefore change the genomic target of the Cas enzyme by simply changing the target sequence...surface-exposed positively-charged grooves on Cas9. gRNA binding induces a conformational change in Cas9, shifting...Fanton, A., Tycko, J., Hinks, M., Chandrasekaran, S. S., Perry, N. T., Schaepe, J., Du, P. P., Lotfy, P., Bassik...approach. Figure 1: Overview of the basic CRISPR mechanism Engineered CRISPR systems contain two components... In this section, we will introduce the basic mechanisms of CRISPR and some of the ways the CRISPR components...efficiency, or flexibility of the Cas enzyme. Basic Mechanisms of CRISPR The requirements for a CRISPR knockout...target DNA. Figure 2: Overview of the NHEJ repair mechanism Cas9 will only cleave a given locus if the gRNA...

...0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette 200–1000...1000 µL single channel pipette Reagents 1X PBS Microcentrifuge tubes Sterile Poly-D-lysine coated coverslips...

... Nonetheless, we recommend performing a buffer exchange before using the purified AAV in vivo . The iodixanol... Workflow Timeline Day 1: Purify Day 2: Buffer exchange and concentration Note: Both steps could be completed...Pool clean fractions. Concentration and buffer exchange: Stocks of Pluronic F68 Pluronic F68, 10% solution...Choi, and R. Jude Samulski. "Production and characterization of adeno-associated viral vectors," (Link ...

...made up of four protein chains: two large heavy chains and two small light chains. These four domains come... producing antibodies, can change the constant region, and therefore change the isotype of antibodies ...: scFv - Single-chain fragment variables (scFvs) contain only the variable heavy chain and variable light... it is determined by a region called the “heavy chain constant region” or Fc. Different isotypes play ...light chain regions of an antibody. They are small enough to be produced in bacteria (~25 kD), but tend... derived from a camelid antibody called a heavy chain antibody (HCAb). Their small size allows them to... conjugation reaction. Many antibodies can be purchased already conjugated, which can save significant...

...Young-Gie Chung, Mi-Yoon Chang, Baek-Soo Han, Sanghyeok Ko, Eungi Yang, Kwang Yul Cha, Robert Lanza, and Kwang-Soo...Plasmids for Stem Cell Research Delivery Method Mechanism Efficiency Safety Pros Cons MMLV-derived Retrovirus...element that transposes via a “cut and paste” mechanism between a vector and chromosomal DNA. PB has a...be removed by re-expressing the transposase. A challenge of using PB to generate iPSCs is that genomic ...the cytoplasm independent of the cell cycle. A challenge of using Sendai is that since it’s replication...clones tested had plasmid integration. Another challenge is that multiple transfection makes it difficult...plasmids due to lower activation of DNA-silencing mechanisms. While minicircles are typically smaller than...

... specific cellular pathways, receptors, or ion channels targeted to specific cell populations (most often...control of neurons through manipulation of ion channels arePharmacologically Selective Actuator Modules...ion pore domain (IPD) to form ligand-gated ion channels (LGICs) that can either excite or inhibit neuronal...activity. Figure 3: PSAM4-based ligand-gated ion channels, their effect, and outcome in neurons. Table 2...opens in a new window) Coward P, Wada HG, Falk MS, Chan SD, Meng F, et al. (1998). Controlling signaling..., Harvey BK, Dannals RF, Pomper MG, Bonci A, Michaelides M (2017). Chemogenetics revealed: DREADD occupancy...Ikefuama EC, Slaviero AN, Silvagnoli AD, Crespo EL, Schalau R, Gott M, Tree MO, Dunbar GL, Rossignol J, Hochgeschwender...

...restriction digest results. Equipment Electrophoresis chamber Pipetman Reagents Liquid DNA aliquot of your plasmid...enzymes. Most companies will have a compatibility chart, such as the (Link opens in a new window) double...in a new window) NEB's compatible cohesive ends chart . If you cannot find compatible sticky ends, you...

... blue mix into a cell counting chamber. Load the cell counting chamber on an automated cell counter and...conical tubes Microcentrifuge tubes Cell counting chamber slide 30 mL luer-lock syringe, BD BD302832 0.2 ...

...uses an electrical field to move the negatively charged DNA through an agarose gel matrix toward a positive...some to the buffer as well. EtBr is positively charged and will run the opposite direction from the DNA...negative, red is positive. The DNA is negatively charged and will run towards the positive electrode. Always...

...numbers of cells minimizes the effects of random chance that could lead to false positive or negative results...being studied. Examples of published selection mechanisms for lentiviral CRISPR libraries include: resistance...that disappear with addition of the selection mechanism Positive Screen In a positive screen, the goal...those cells that do not survive the selection mechanism. You’ll infect two sets of cells and subject one...consult these links: NGS Company Resources Science Exchange Scientist (formally Assay Depot) Resources Find...

...Quality Control (QC) Pooled libraries can be challenging and expensive to ensure adequate quality, but...can be used but this dramatically increases the chances of individual plasmids being lost from the pool...taken to perform adequate NGS based QC to ensure no change in representation compared to the pre-amplified...

...neighboring genes. The short coding sequences of channelrhodopsins, halorhodopsins, and other optogenetic genes...have thus far been identified, with the best characterized and most commonly used being AAV2. These serotypes...preferentially transducing specific cell types. The chart below gives a summary of the tropism of AAV serotypes...Many of these hybrid viruses have been well characterized and may be preferred over standard viruses for...viral vectors for enhanced gene transfer. Kwon I, Schaffer DV. Pharm Res. 2008 Mar;25(3):489-99. PubMed ....

... Rev Envelope Plasmid Interchangeable: usually encodes for VSV-G Interchangeable: usually encodes for ...packaging plasmid(s) one envelope plasmid After media change and a brief incubation period, supernatant containing... sites. For more information about cloning and changing a MCS, visit Addgene's Plasmid Reference and Protocol... Petit C, Guetard D, Nerhbass U, Montagnier L, Charneau P. Cell. 2000. 101(2): 173-185. PubMed . Woodchuck...

...Iodixanol Gradient Ultracentrifugation Protocol Mulitchannel Pipetting Technique Learn how to pipette multiple...multiple samples at once using a multichannel pipette and avoid inconsistent pipetting with our simple ...