We narrowed to 452 results for: proc.2

...Workflow Timeline Day 0: Seed and transduce cells Day 2: Replace media with fresh media containing selection...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container...the appropriate antibiotic. Incubate the cells for ~2 weeks. All of the cells in the untransduced (negative...disturb the colonies. Count the colonies for at least 2 of the dilutions. Pro-Tip The higher dilution wells...stained with 0.1% crystal violet and counted. Figure 2: A549 cells were transduced with the indicated serial...transduction, and not during the cell seeding step. Procedure Before beginning a colony formation assay, the...

...intact. First-generation plasmids include (Figure 2): Transfer plasmid — contains transgene and wild-type...envelope removed Envelope plasmid — contains env Figure 2: First-generation lentiviral plasmids. Created with...plasmid 1 — contains gag and pol Packaging plasmid 2 — contains rev Envelope plasmid — contains env Third-generation...The inside out of lentiviral vectors. Viruses , 3 (2), 132–159. https://doi.org/10.3390/v3020132 PMID: ...Infectivity and Integration Targeting. PLoS ONE , 2 (12), e1340. https://doi.org/10.1371/journal.pone.0001340...features of lentiviral vectors. Human Gene Therapy , 24 (2), 132–142. https://doi.org/10.1089/hum.2012.229 PMID...lentiviral vectors from your plasmids (Figure 5). This process requires a production cell line, typically human...

...RNAzol®, QIAzol® (for Protocol Option #2) Water-saturated Phenol 2 M Sodium Acetate pH 4 Chloroform/Isoamyl....5% (wt/vol) N-laurosylsarcosine (Sarkosyl) 0.1 M 2-mercaptoethanol TRIzol® or similar product such as...recipe). If using TRIzol®, jump down to the Option #2 - TRIzol® Protocol section below. Homogenize or lyse...following sequentially to 1 mL of lysate: Add 0.1 mL of 2 M sodium acetate (pH 4.0), mix thoroughly by inversion...aliquots of it and storing those in -80°C. Option #2 - TRIzol® Protocol Homogenize or lyse tissues or cells...by hand for 10 seconds. Incubate the sample(s) for 2-3 minutes on ice and centrifuge for 15 minutes at ...with the volatile reagents in the list above. Procedure Option #1 - Solution D Protocol Before starting...

...temperature between 50-60°C for your PCR primers. Step 2: Linearize Vector In this example, the vector is cut...20-30 Eluted DNA 10-50 ng/μl 1 dGTP (100mM) 2.5 mM 2 DTT (100 mM) 5 mM 1 BSA (10 μg/μl) 0.25 μg/μl 1 T4...your treated vector and insert at a molar ratio of 1:2 or 1:3, using between 20 and 50 ng of vector per annealing... reaction is now ready for transformation. Use 1-2 μl of annealing reaction for each transformation, ...together through the transformation/replication process. LIC employs long overhangs to form a stable association...free nucleotides from your PCR product before proceeding, as they will interfere with the exonuclease ...

...agarose concentrations (e.g., 2 g of agarose in 100 mL of TAE will make a 2% gel). Mix agarose powder with...gels are commonly used in concentrations of 0.7% to 2% depending on the size of bands needed to be separated...concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr binds...allows you to gauge how far the DNA has migrated; 2) it contains a high percentage of glycerol that increases...Introduction Gel electrophoresis is the standard lab procedure for separating DNA by size (e.g., length in base...Gel loading buffer, New England Biolabs B7022S Procedure Pouring a Standard 1% Agarose Gel: Measure 1 g... from Your Gel: If you are conducting certain procedures, such as molecular cloning, you will need to ...

...each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30-60 ...cells you are using). Put the tubes back on ice for 2 min. Add 250-1,000 μl LB or SOC media (without antibiotic...instead of on ice Reduce step 4 from 20 - 30 mins to 2 mins on ice before heat-shock Shorten or skip the ...DNA Ligation Introduction Transformation is the process by which foreign DNA is introduced into a cell.... competent cells. This is a relatively simple procedure and is useful for performing low efficiency transformations...media Competent cells DNA you'd like to transform Procedure Take competent cells out of -80°C and thaw on ...competent cells) to ensure that your transformation procedure is working. TIP: Sometimes less is more. Although...

...Day 1: Seed individual cells in a 96-well plate Day 2–14: Monitor cells for growth and expand cells Day ...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...channel pipette 200–1000 µL single channel pipette CO 2 incubator Pipet controller Hazardous waste container...approximately 50–60% confluent. For 293T cells this is about 2 × 10 6 cells in a 10 cm dish. Each 10 cm dish should...the highest or lowest transgene expression ( Figure 2 ). Sample Data Figure 1: Generation of monoclonal ...inferred by the differences in colony size. Figure 2: Cas9 expression in monoclonal cell lines generated...for different cell types. Before starting this procedure, be sure that the cell pool has been sufficiently...

...BSL-1 and BSL-2 Labs Learn how to best protect yourself when working in BSL-1 and BSL-2 labs Watch the...Two (BSL-1 and BSL-2) Safety measures for laboratories operating at BSL-1 and BSL-2 Watch the Video! Water...the building blocks for many more complicated procedures. Name Description (Link opens in a new window...

...Workflow Timeline Day 0: Seed and transduce Cells Day 2–3 (am): Remove media, replace with fresh media containing...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container...cell death, the cell media should be changed every 2–3 days to maintain the dose of antibiotic, which may...confluent 10 cm dish can be expanded into two 75 cm 2 flasks, etc. Pro-Tip This selection method results... be subjected to multiple freeze-thaw cycles. Procedure Before beginning, determine the optimal dose of...antibiotic. This is the beginning of the selection process, which will begin the selection of a stable cell...

... recover, set up overnight growth (Estimated time 2-3 hours) Transformation should be performed at the... day to ensure that growth times are limited. Day 2: Harvest cells and purify DNA (Estimated time 3-4 ... Tubes should contain a total of 5 mL (3 mL SOC + 2 mL transformed Endura from two separate transformations...has been absorbed by the agar. This usually takes 1-2 minutes. Critical Be careful not to rip or shred the...incubation if needed. Place 100 mL sterile LB at 4 ℃. Day 2 (morning) Before beginning, prechill at least four...pellet. The total weight of each pellet should be ~1-2 g. Pro-Tip Make sure to weigh the empty tube beforehand...Libraries Molecular Biology Reference How do I process my Addgene pooled library? Introduction Please ...

...cooling to room temperature (~45 minutes). Method #2 Place mixed oligos in a PCR tube. Place tube in a ...a thermocycler programmed to start at 95°C for 2 minutes. Then, gradually cool to 25°C over 45 minutes...vector with 0.75-6 ng of annealed oligos). Transform 2-3μL into your favorite competent bacteria and plate...to add short tags or shRNAs to any vector (the procedure will simply differ in terms of primer design)....be performed enzymatically later. Experimental Procedure Digest and purify vector While waiting for your...

...Fluorescent microscope 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... plate 4% Paraformaldehyde 5 mg/mL 4′,6-diamidino-2-phenylindole (DAPI) Bovine serum albumin (BSA) Triton...grow to the desired density before labeling. Section 2: Fixing and permeabilizing cells Gently aspirate the...of the diluted antibody to the wells and incubate 2 h at RT . Remove the primary antibody and dispose ...stock solution into 5 mL PBS. Protect from light. Procedure Section 1: Seeding cells Place a sterile poly-...

...components, see the AAV Plasmid Elements table . Figure 2: Plasmids required for rAAV vector production. Created... pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism...the central nervous system . Molecular Therapy, 10 (2), 302–317. https://doi.org/10.1016/j.ymthe.2004.05.024...the production of AAV vectors . Microorganisms, 12 (2), 384. https://doi.org/10.3390/microorganisms12020384...retrograde access to projection neurons . Neuron, 92 (2), 372–382. https://doi.org/10.1016/j.neuron.2016.09.021...genome. Instead, the linear rAAV DNA is typically processed into a double-stranded circular episome which ...AAV serotypes. Pseudotypes Pseudotyping is the process of producing viral vectors in combination with ...

... plate. Incubate plates at 37 ℃ for 18 hours. Day 2 Observe plates for colony formation. Shown below are...individual colonies and effective selection. 150 µL of 2 mg/mL Carbenicillin plated over-agar Plate shows less...individual colonies with smaller size than the 1 mg/mL and 2 mg/mL plates and effective selection. Selection Curve...resistance gene (or other antibiotic resistance) Procedure Day 1 Prepare carbenicillin to a concentration...

...Browse Plasmids: Double-Strand Break (Cut) Figure 2: Overview of the NHEJ repair mechanism. Multiplex ...systems enable researchers to target anywhere from 2–7 genetic loci by cloning multiple gRNAs into a single...Cas9 is included in the gRNA-containing plasmid, or 2-vector systems, in which Cas9 must be delivered separately...your experimental cell population (Figure 8E). In a 2-vector system, you’ll need to either co-infect with...the presence of infectious organisms (like SARS-CoV-2 ) and genetic mutations. Similar to Cas9 and Cas12...Cas enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme...off-target effects by using a single Cas9 nickase and 2 different gRNAs, which bind in close proximity on ...

...cells; function is unknown Monomer with a valency of 2 IgE Protects against parasites and is responsible ...such as anaphylactic shock Monomer with a valency of 2 IgG The most abundant isotype in blood, also found...secondary immune response Monomer with a valency of 2 IgG1; IgG2; IgG3; IgG4 IgM Produced during the primary... ranging from antibody fragments (shown in Figure 2) to completely unrelated molecules. Examples include...used for assembling protein nanostructures. Figure 2: An antibody (IgG, far left) compared to alternative...randomized reorganization process, creating a unique binding site. This process, known as V(D)J recombination...delaying a process through sequential rounds of validation. Validation of an antibody is a process separate...

...components (Figure 2), and the optimal system will likely depend on your cell type. Figure 2: Different delivery...either (1) a lack of gRNA and/or Cas9 expression or (2) a lack of efficient target cleavage in cells that...CRISPR-Based genome editing. Trends in Biotechnology , 36 (2), 173–185. https://doi.org/10.1016/j.tibtech.2017.11.006...genetic manipulation to model a specific disease or process of interest. Do you want to: Generate complete ... method of delivery and the cell type. Before proceeding, we recommend asking labmates/colleagues, searching... genes modulate transcription and nascent RNA processing. Nature Structural & Molecular Biology , 30 (...

...s) Buffer BSA (if recommended by manufacturer) dH 2 O up to total volume Pro-Tips The amount of DNA that... 10x Buffer 3 µL 10x BSA (if recommended) x µL dH 2 O (to bring total volume to 30µL) The amount of restriction...from 45 mins to overnight. For diagnostic digests, 1-2 hours is often sufficient. For digests with >1 µg ...loading dye Electrophoresis buffer Pipet tips Procedure Select restriction enzymes to digest your plasmid...

...types: positive screens and negative screens (Figure 2). Both types of screen employ a selection method relevant...planning to conduct a pooled library screen. Figure 2: A simplified outline for using lentiviral gRNA libraries...for amplifying and using a pooled library. The process diverges for lentiviral of DNA-based screens. NGS...positive and negative screens follow the same basic process: Amplify the library (electroporation and maxiprep...

...streak #2. Using a third sterile pipette tip, toothpick, or sterilized loop, drag through streak #2 and spread... (with appropriate antibiotic) Bacterial stab Procedure Obtain an LB agar plate with appropriate antibiotic...colonies. Once you have single colonies, you can proceed to Recovering Plasmid DNA or use the individual...