...lines of interest Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette...CI) Heat-inactivated FBS 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect...selective medium is used, it may have additional biological effects on the cell line. In the absence of selective... medium throughout experiments be determined empirically, and that the cell population be monitored regularly...individual cells by passing several times through a serological pipet or by passing through a 40 µm cell strainer...will be used to seed the 96-well plate. Sample Calculation First, use a hemocytometer to quantitate the ...well plate, make 10 mL of a 5 cell/mL solution. Calculate the total cells needed: Total cells needed: 10...

...for short term studies of protein function. We specifically use this protocol with a subclone of HEK293T...perform your experiment Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette... or sodium hydroxide to adjust the pH to 7.0. Typically, the solution will be basic and will need adjustment...Before You Start The health of the cell line is critical for obtaining high levels of virus. HEK293T cells...optimal mass DNA:mass PEI ratio will need to be empirically determined for each new batch of 1 mg/mL PEI ...batches of PEI depending on the user, quantities of chemical used, volumes, pH adjustment etc. Consequently...Pro-Tip The ratio of µg DNA:µg PEI needs to be empirically determined. Once a batch of PEI is prepared, ...

...backbone of the two DNA fragments. This reaction, called ligation, is performed by the T4 DNA ligase enzyme...nucleotides together. After ligation, the insert DNA is physically attached to the backbone and the complete plasmid...digestion . Most restriction enzymes digest DNA asymmetrically across their recognition sequence, which results...digested end of the DNA fragment. The overhangs, called "sticky ends", are what allow the vector and insert.... Use a (Link opens in a new window) ligation calculator to easily quantify how much vector and insert...of DNA, buffer and ligase into a 10μL reaction, scale the reaction size as necessary - being sure to increase...vector end. (Link opens in a new window) Ligation calculators are easily found on the web. Just enter the concentration...

...flasks). Cell stacks provide an efficient means to scale-up without having to handle a large number of T-...am): Harvest cells Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette...Thermo Fisher, 12605010) 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect...Before You Start The health of the HEK293T cells is critical for optimal AAV yield. Do not overgrow your cells...incubator for 24 h–36 h. Proceed with transfection: Calculate the amount of each plasmid needed to have a 1:... Therefore, for each plasmid we need: Sample Calculations RepCap: 0.08 μg/bp × 7,265 bp = 582.1 μg Volume...detach easily. Transfer cells and media into 500 mL conical vessels. Rinse CS5 once with 100 mL of PBS and ...

...gene. For example, the Whitehead Institute for Biomedical Research hosts an siRNA Selection Program that... #A9414 Luria Broth Agar (LB agar) American Bioanalytical: #AB01200-02000 Ampicillin VWR: #7177-48-2. ...transfection in a 6 cm plate. The protocol can be scaled to produce different amounts of virus as needed... transfection reagent in serum-free OPTI-MEM. Calculate the amount of Fugene® and OPTI-MEM necessary given... Sigma-Aldrich: #H9268 Protamine Sulfate* MP Biomedicals: #194729 Puromycin* Sigma-Aldrich: #P8833 Target...0.5 mL for a high MOI, and 0.1 mL for a low MOI). Scale the amount of virus added depending on the size ...antibiotic Per 40 grams of powder from American Bioanalytical catalog # AB01200-02000, LB contains: 10g tryptone...

...resistant colonies Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette...mL), Millipore TR-1003-G 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect...tissue culture treated dish, Corning 3516 15 mL conical tubes, VWR 21008-216 Lentivirus preparation Reagent...You Start The health of the target cell line is critical for obtaining accurate titers. Check the cells...required to kill your target cell line needs to be empirically determined. Treat the target cells with a range... can usually be omitted because this dilution typically produces a lawn of cells, which cannot be accurately...integration events decreases as the dilution increases. Calculate the transduction units per mL (TU/mL) as follows...

...analyzing the gene’s function. The process is shown graphically in the following cartoon, in which we are adding...TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3’) into this calculator we get a final Reverse Primer sequence of 5’-...reaction and 1μg of recipient plasmid. It is also critical that as much of the recipient plasmid as possible...step, depending on the phosphatase you choose. CIP (calf alkaline phosphatase) or SAP (shrimp alkaline phosphatase...base pairs for each varies, it is difficult to calculate this based on DNA concentration alone. One method...electro-competent cells instead of the more common chemically-competent cells. The number of bacterial colonies...will tell you your “background” level or more specifically it will tell you how many colonies you can expect...

...: Buffer exchange Equipment Class II, Type A2 Biological Safety Cabinet 4 °C Refrigerator Pipette controller...controller Benchtop centrifuge compatible with 50 mL conical tubes NanoDrop spectrophotometer 37 °C, 5% CO 2...VWR 89130-900 50 mL pipette, VWR 89130-902 50 mL conical tubes, VWR 89039-656 Microcentrifuge tubes, VWR...inhibitors to 1X if this has not already been done. We typically start with about 250 mL of supernatant and add...buffer. re Collect the flow-through in a 50 mL conical tube and dispose after use. Carefully pour the ...closure and loosen cap. Place column in a 50 mL conical collection tube. Centrifuge column at 1000 x g ... to reach the desired concentration. Pro-Tip Periodically check the concentration on the NanoDrop Spectrophotometer...

...Stack such that the electrical contacts are aligned with the corresponding electrical contacts on the blotting... 37 °C . Determine the absorbance at 590 nm . Calculate the average absorbance of the duplicate samples...vary between samples and target proteins but is typically between 10–50 µg . If the protein is in low abundance...Bottom Stack on the blotting surface. Align the electrical contacts on the blotting surface of the iBlot...concentration will vary between antibodies but is typically between 1–10 μg/mL. Incubate the membrane overnight...concentration will vary between antibodies but is typically between 1–10 μg/mL. Incubate the membrane with...

...primary antibody to use will vary and needs to be empirically determined. If possible, run an initial test ...Microplate shaker ELISA plate washer (optional) Scale Microsoft Excel or similar software Reagents Purified...Bovine serum albumin (BSA), VWR 10273-264 50 mL conical tubes, VWR 89039-656 96-well polyester (clear) ...antibody concentration will vary and needs to be empirically determined. We suggest starting with three concentrations... rpm for 15–30 min . Pro-Tip Check the plate periodically to determine when the desired color change has...curve will vary between targets and needs to be empirically determined. The dilution series created in section...don’t forget to factor in that dilution when calculating the original sample concentration!). Sample Data...

...recommend 5’ and 3’ sequencing primers. These primers typically anneal to the backbone and can help you verify...sequencing results as a trace file (.ab1) which graphically depicts the sequence as a series of colored peaks...at the trace file, you will see that these base calls are unreliable. This is an example of a trace file...apparent mismatch/mutation may be the result of a mis-called peak in the trace file. If the mutation is not...and Chromas (PC). How can I tell if a peak is mis-called? Open the trace file and use the search feature...Check the trace file and see if you can manually call the correct base at the position. Sometimes an “...

...after working in the lab. Ensure that a designated chemical waste accumulation site is present, and that you...before starting the work. Before working with chemicals, first review their material safety data sheets...waste that may be contaminated with pathogens or chemicals that present a danger to people and the environment...disposal, commonly done with an autoclave. Only mechanical pipetting should be done in the laboratory. Don... evacuate and close off the area and alert the chemical hygiene officer. BSL-2 Guidelines Remember, the...so at your own risk; you should ensure that any local guidance is also adhered to. None of the authors...

...treatment of your recipient plasmid as well as a specifically designed test digest later to verify that the...plasmid and 1μg of recipient plasmid. It is also critical that as much of the recipient plasmid as possible...step, depending on the phosphatase you choose. CIP (calf alkaline phosphatase) or SAP (shrimp alkaline phosphatase...base pairs for each varies, it is difficult to calculate this based on DNA concentration alone. One method...electro-competent cells instead of the more common chemically-competent cells. The number of bacterial colonies...will tell you your “background” level or more specifically it will tell you how many colonies you can expect...

...Adeno-associated viral vectors (AAV). This protocol specifically uses primers and probes targeting the ITR elements... is genome copies and generally represents the physical number of viral particles. Users may need to run...biosafety regulations. Equipment Class II, Type A2 Biological Safety Cabinet, Labconco 302411100 Droplet Digital...cartridge holder with bleach and place in the Biological Safety Cabinet (BSC). Make sure that the BSC ...Pro-Tip Prepare the viral dilutions in a separate biological safety cabinet (dilution BSC) than that used ... a CSV file and use the following equation to calculate the titer for each sample: $$T = {R*C*D*1000\over...

...material, use an analytical scale. This will provide more accuracy as these types of scales display more ...are weighing by looking for a weight range on the scale. Make sure that the weight of the material you’re... balance. This will help you remove it from the scale later on as otherwise, the paper could stick to ...

...larger conical tubes. Depending on the centrifuge their speed ranges can vary, but are typically lower ... the two, use a conversion calculator or chart like the G Force calculator (Link opens in a new window...Tabletop Centrifuges sit on your benchtop and typically hold the common 1.5 mL microfuge tubes used in...

... Addgene Protocols On this page you'll find practical lab protocols that you can use for a wide range...Generating Stable Cell Lines with Lentivirus Genomically integrate your DNA sequence into a cell line ... SYBR Green Technology Use qPCR to measure the physical titer of your adeno-associated virus preparation...AAV Titration by ddPCR Use ddPCR to measure the physical titer of your adeno-associated virus preparation...Lentivirus Titration by ddPCR Use ddPCR to measure the physical titer of your lentivirus preparation Antibodies...

...depends on your application. A diagnostic digest typically involves ∼500 ng of DNA, while molecular cloning...largely determined by the volume of DNA to be cut. A typical restriction digestion reaction could look like ...reaction in 1 hour. Using this ratio, you can calculate the minimal amount of enzyme for your reaction...the vector if you are cloning in an insert. CIP (calf alkaline phosphatase) or SAP (shrimp alkaline phosphatase...enzymes cut sequences which are similar, but not identical, to their recognition sites. This is due to "...

...and optical switches Sensors are genetically-encoded reporters of molecular signals; e.g., calcium indicators...Deisseroth K. 2005. Millisecond-timescale, genetically targeted optical control of neural activity. Nat... utilized to control the localization of a protein of interest. A genetically-encoded nuclear Cry2 fusion...MJ. 2006. Next-generation optical technologies for illuminating genetically targeted brain circuits. J...17375185 Kim B, Lin MZ. 2013. Optobiology: optical control of biological processes via protein engineering. Biochem... including explanations of microbial opsin and optical switch terminology. Science...Optogenetics Guide Introduction Microbial Opsins Optical Switches References Introduction The field of optogenetics...

...antibody’s constant region - specifically, it is determined by a region called the “heavy chain constant ...reagents are molecular tools used in the lab to specifically and tightly bind to other proteins. Antibodies...interest. The protein that an antibody recognizes is called an antigen, while the specific binding site is ...referred to as an epitope. Production Antibodies are typically produced in one of three different methods: in...independently manipulated. Antibodies produced this way are called recombinant antibodies. Recombinant antibodies ...are responsible for binding the antigen, and are called the antigen binding fragments (Fab). The tips of...the Y structure and the lower part of the arms is called the constant region, and is shown in dark blue ...

...significantly and should be empirically determined for your sample. Typical ratios may range from 1:1 to...pipettes Benchtop centrifuge compatible with 50 mL conical tubes Automated cell counter 37 °C, 5% CO 2 incubator...antibody plasmid DNA 500 mL vented flask 50 mL conical tubes Microcentrifuge tubes Cell counting chamber.... Transfer the HEK293 cells and media to 50 mL conical tubes. Centrifuge for 15 min at 3100 x g to pellet...

...polymerase that can withstand radical temperature changes during a typical PCR. The DNA polymerase has ...template), a researcher can create thousands of identical copies using a simple set of reagents and a basic...GC-rich templates. What does each ingredient specifically do? Template DNA: Contains the portion of DNA...Buffer with MgCl 2 : Provides an optimal and stable chemical environment for the DNA polymerase to work adequately...

... of identical bases. Browse Plasmids: Prime Edit Large Scale Edits CRISPR can generate large-scale genetic...takes place during the S and G2 phases. Chemically or genetically inhibiting genes involved in NHEJ or upregulating...properties, often called a reeling mechanism. The targeted strand is cleaved non-specifically by Cas3 along...immunoprecipitation (enChIP) using CRISPR. Biochemical and Biophysical Research Communications . 439 (1), 132... generating gRNAs makes CRISPR one of the most scalable genome editing technologies, making CRISPR perfect...gRNAs will be expressed in the same cell. This is called multiplexing , and increases the likelihood that...DNA with no homology elsewhere in the genome. Realistically, most gRNA targeting sequences will have additional...

...responded to a clinically approved drug, the PSAM domain was engineered to respond specifically to the anti-smoking...coelenterazine (CTZ) or chemical variations thereof (hCTZ), or furimazine (Fz) or chemical variations thereof...integrate opto- and chemogenetics by using physical and biological light sources for opsin activation. Proc...unique and not a common property of either typical or atypical antipsychotic drugs: is M1 agonism a pre-...Inhibitory luminopsins: genetically-encoded bioluminescent opsins for versatile, scalable, and hardware-independent...been shown to be involved in a wide variety of biological processes, including initiating signaling pathways...ligands, could be activated by synthetic ligands. Specifically, Receptors Activated Solely by Synthetic Ligands...

...stable cell lines Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette...mL), Millipore TR-1003-G 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect...tissue culture treated dish, Corning 3516 15 mL conical tubes, VWR 21008-216 Lentivirus preparation Appropriate...You Start The health of the target cell line is critical for obtaining accurate results. Check the cells...and/or harvest to test for protein expression. Typically, cells transduced with lower dilutions of the ...

...make bacteria more susceptible to either chemical or electrical based transformation, generating what are...antibiotic containing agar plate. This step is not critical for Ampicillin resistance but is much more important...large plasmids (>10 kb) or BACs, what can I do? Chemically competent cells are fast and easy to use, but...

...should have identical sequences on the ends (sequences A and B in the figures). These identical sequences...PCR with primers that contain a 5′ end that is identical to an adjacent segment and a 3′ end that anneals..., Hutchison CA, Venter JC, Merryman C. (2010). Chemical synthesis of the mouse mitochondrial genome. Nature...

...visualization and purification. Electrophoresis uses an electrical field to move the negatively charged DNA through...eye protection and gloves when working with this chemical. Note: If you add EtBr to your gel, you will also...approximately 75-80% of the way down the gel. A typical run time is about 1-1.5 hours, depending on the...polyacrylamide gel electrophoresis (PAGE), which is typically used to separate 5 - 500 bp fragments. How do ...

...purification of cellular components, such as DNA, but practically this isolation is only feasible for relatively...Plasmids are fragments of double-stranded DNA that typically carry genes and can replicate independently from...and eukaryotes, they play the most significant biological role in bacteria where they can be passed from...to the field of recombinant DNA, in which many identical DNA fragments can be generated. The combination... 6000 basepairs) to enable easy manipulation. Typically plasmids contain the minimum essential DNA sequences... DNA, and this process of generating multiple identical copies of a recombinant DNA molecule is known ...manipulate genes and other genetic elements using specifically engineered plasmids, commonly referred to as...

...medium. This dot is composed of millions of genetically identical bacteria that arose from a single bacterium...draw in discontinuous lines. Start by streaking a vertical line of bacteria along one edge of the plate. ...

...100-1000 base pairs long and are adjacent and typically upstream (5’) of the sense or coding strand of...transcribed by the RNA polymerase. DNA sequences called response elements are located within promoter regions... sequence thought to be functionally equivalent called the Pribnow box which usually consists of the six...Promoter The final portion of the promoter region is called the distal promoter which is upstream of the proximal...back on itself which helps to explain how many physically distant DNA sequences can affect transcription... -35 position, simply titled the -35 element, typically consists of the sequence TTGACA and this element... operator. Operons are common in prokayotes, specifically bacteria, but have also been discovered in eukaryotes...

... watching, and we hope you found the video radd-ically helpful!    More resources at Addgene Addgene'...

...polymerase can create overhangs of varying length (typically 10-12 bp) based on a specific sequence. The annealed... the replication cycle. Empty vectors for LIC typically employ type II restriction enzymes (e.g. BsaI)...back the guanosine residue and become stalled. A typical T4 Pol reaction is shown. Mix the components (...

...share quick tips to help you master fundamental technical lab skills, like pipetting! We’ll release a new...

...edition of our Viral Vectors 101 eBook!  This practical guide to using viral vectors in the lab is a helpful...

...Mackin Lab from University of Colorado -Anschutz Medical Campus (@CSD_CUAMC) caught a case of holiday cheer...

...lab! Introduction Microscopes are emblematic of biological research and are found in many different types...suggests, light microscopes take advantage of the physical properties of light to detect small objects. Two...the light follows from the source to your eyes is called the light path . Light travels from the light source...

...your life a little easier. These can range from technical tips as in our episode on Barcoding, to basic ...

...software engineers, surprisingly, who have deep technical knowledge but little biology experience.) The ...

... depositing paper for more information Edward Callaway   For researchers in countries with applicable...