We narrowed to 492 results for: ARA-2

...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...cloning, and titering and testing your viral preparations. Addgene...Learn about selecting and using a centrifuge to separate different components in a liquid sample Using ...restriction enzymes Agarose Gel Electrophoresis Separate DNA by size on an agarose gel Watch the Video!...Protocols for titering and testing your virus preparations. Name Description Link to Video General Transfection...Ultracentrifugation Purify adeno-associated virus from a preparation produced in mammalian cells Watch the Video! ...

... solution can be stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working... transfected using 1:1, 1:2, 1:3 and 1:6 µg of pRosetta :µg of PEI. The 1:2 and 1:3 ratios provided high... for viral production) Day 1: Transfect Cells Day 2 (am): 18 h post transfection - Remove media, replace...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... use. Thawed aliquots should be discarded after 1–2 months. 1 mg/mL polyethylenimine, linear MW 25,000...times a week: Monday: Plate 1x10 6 cells in a 75 cm 2 flask in a volume of 15 mL. Wednesday: Plate 1x10 ...

...agarose concentrations (e.g., 2 g of agarose in 100 mL of TAE will make a 2% gel). Mix agarose powder with...concentrations of 0.7% to 2% depending on the size of bands needed to be separated - see FAQs below . Simply...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... typically used to separate 5 - 500 bp fragments. How do you get better separation of bands? If you have...electrophoresis, including tips to improve resolution and separation of bands. Protocols...electrophoresis is the standard lab procedure for separating DNA by size (e.g., length in base pairs) for ...

...RNAzol®, QIAzol® (for Protocol Option #2) Water-saturated Phenol 2 M Sodium Acetate pH 4 Chloroform/Isoamyl...sample(s) for 2-3 minutes on ice and centrifuge for 15 minutes at 12,000 × g at 4°C to separate RNA from the....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...sample(s) for 15 minutes at 12,000 × g at 4°C to separate RNA from the rest of the tissue/cell lysate. Pro-Tip...

...materials D.2 Screening for inserts E. Producing Lentiviral Particles E.1 Recommended materials E.2 Protocol...Published articles H.2 Web resources I. Appendix I.1 Sequence of pLKO.1 TRC-Cloning Vector I.2 Recipes I.3 Warranty...VWR: #7177-48-2. Use at 100 μg/mL. Carbenicillin VWR: #80030-956. Use at 100 μg/mL. C.2 Annealing Oligos...oligo 5 μL Reverse oligo 5 μL 10x NEB buffer 2 35 μL ddH 2 O Incubate for 4 minutes at 95°C in a PCR machine...buffer 1 1 μL AgeI add ddH 2 O to bring to 50 μL final volume Incubate at 37°C for 2 hours. Purify with Qiaquick...buffer for EcoRI 1 μL EcoRI 14 μL ddH 2 O Incubate at 37°C for 2 hours. Run digested DNA on 0.8% low melting... For a standard T4 ligation, mix: 2 μL annealed oligo from step C.2 20 ng digested pLKO.1 TRC-cloning ...

...Warnings HEK293 cells are considered biosafety level 2. Please ensure that you are in compliance with your... 18, 2022 Workflow Timeline Day 1: Seed cells Day 2: Transfect cells Day 3-6: Feed cells Day 7: Harvest...conical tubes Automated cell counter 37 °C, 5% CO 2 incubator with shaking platform set to 120 rpm 37 ...250 mg Benzamidine 25 mL Aprotinin saline solution (2 mg/mL) Mix well and sterilize through a 0.2 µm PES... a 500 mL vented flask.Incubate in a 37 °C, 5% CO 2 incubator on a shaking platform set to 120 rpm. Pro-Tip... not use cells that are over 30 passages. Section 2: Transfection Check the cell density and viability...the culture. Pro-Tip Culture should be between 1.5–2 x 10 6 cells/mL with >95% viability to proceed with...

... solution can be stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working...μg total DNA to μg PEI ratios of 1:1, 1:2, 1:3 and 1:6. The 1:2 and 1:3 total DNA:PEI μg ratios provided...packaging cells Day 1 (pm): Transfect packaging cells Day 2 (am): 18 h post-transfection. Remove media, replace...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...culture plates. Incubate the cells at 37 °C, 5% CO 2 for ~20 h. Prepare a mixture of the three transfection...plasmid using a variety of ratios. Check the cells 1-2 days after transfection to determine what ratio gives...

...coverslips HeLa cells 24-well plate 4% Paraformaldehyde 5 mg/mL 4′,6-diamidino-2-phenylindole (DAPI) Bovine serum...Fluorescent microscope 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...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 ... of cold 4% paraformaldehyde in PBS on ice for 15 min . Pro-Tip While 4% Paraformaldehyde fixation works...primary antibodies raised in a mouse. Reagent Preparation Permeabilization buffer: Dilute 20 µL of Triton...be better for some applications. Remove the paraformaldehyde and follow your institution's laboratory safety...

...mL glass bottle: 4 g NaCl 4 g Tryptone 2 g Yeast Extract and dH 2 O to 400 mL Note: If your lab has pre-mixed...start 2 mL in a Falcon tube. For larger preps, you might want to use as much as a liter of LB in a 2 L Erlenmeyer...indicated, the antibiotic powder can be dissolved in dH 2 0. Addgene recommends making 1000X stock solutions...individual plasmid within a single bacterial cell (Figure 2). Large plasmids usually have a low copy number (approximately...determine if your plasmid is high or low copy. Figure 2: High versus low copy plasmids in bacteria. Created...After incubation, check for growth, which is characterized by a cloudy haze in the media (Figure 1). Notes...

...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, ...“stuffer” sequence allows for electrophoretic separation of linearized vector from the reaction mixture...After the digest is complete, you will need to separate the linearized vector from the reaction mixture...

...module_attribute "schema_version" is_json="true" %}{% raw %}2{% endraw %}{% end_module_attribute %}{% module_attribute...technology, and the user may need to optimize numerous parameters to generate the desired edit. The wrench: Gene...

...ask new and interesting questions in your own lab. 2. Seek multiple mentors during your postdoc At the ...corresponding author publications (published, not in preparation) A history of successful funding (fellowship ...

...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...15 mL conical tubes, VWR 21008-216 Lentivirus preparation Appropriate antibiotic for selection (e.g. puromycin...puromycin, blasticidin) Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (...

...contain a total of 5 mL (3 mL SOC + 2 mL transformed Endura from two separate transformations). Pro-Tip Do not... 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 ...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...Falcon Conical tubes (Fisher, 14-432-22) Reagent Preparation Prepare, sterilize, and pour all LB Agar + Antibiotic...

... 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 genes, as one does not need to prepare separate batches of antibiotic-containing agar. This protocol...

...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...culture dish, Corning 430167 (optional) Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-...

...-hsdRMS-mcrBC) Phi80lacZΔM15 Δ-lacX74 recA1 araΔ139 D(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG tonA...mrr-hsdRMS-mcrBC) Φ80dlacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ- rpsL (StrR) nupG trfA...+B+ lacIq ∆(lacZ)M15 zzf::Tn10 (TetR) ∆(ara-leu) 7697 araD139 fhuA ∆lacX74 galK16 galE15 e14- Φ80dlacZ...mrr-hsdRMS-mcrBC) Phi80lacZM15 Δ-lacX74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG Common...Invitrogen F- mcrB mrr hsdS20 (rB-, mB-) recA13 supE44 ara-14 galK2 lacY1 proA2 rpsL20 (StrR ) xyl-5 λ- leu ...unique restriction enzyme recognition site (Figure 2). These elements allow for the propagation of the ...elements are summarized in the table below. Figure 2: General structure of a plasmid. Plasmid Element Description...

...Cryptochrome 2 and CIB1 Blue-light–mediated induction of protein interactions based on Arabidopsis thaliana... OPTOSTIM PHR domain2 of cryptochrome 2 (Cry2) from Arabidopsis thaliana is fused to truncated forms of... derived from direct comparative analysis of microbial opsins. Nat Methods, 9 (2), 159–172. https://doi.org...influx into the cell when stimulated. Channelrhodopsin-2 (ChR2), the first widely adopted optogenetic tool,...thaliana cryptochrome 2 and CIB1 450 LARIAT Inhibits protein function by reversibly sequestering targets into...customizable TALE DNA-binding domain with cryptochrome 2 protein and its interacting partner CIB1 to control...control of biological systems. Nat Rev Methods Primers, 2 , 55. https://doi.org/10.1038/s43586-022-00136-4 PMID...

...use of pAdEasy-2 (Addgene #16401) can increase the capacity of the rAdV vector. pAdEasy-2 does not contain... incorporate additional genomic deletions. Figure 2: First-generation rAdV vectors. Created with BioRender.com...and have to be handled at biosafety level two (BSL-2). They are highly immunogenic and can trigger a strong...era of personalized medicine . Genes & Diseases, 4 (2), 43–63. https://doi.org/10.1016/j.gendis.2017.04.001...adenoviruses using the AdEasy system . Nature Protocols, 2 (5), 1236–1247. https://doi.org/10.1038/nprot.2007.135...Adenoviral vector vaccine platforms in the SARS-CoV-2 pandemic . NPJ Vaccines, 6 (1), 97. https://doi.org...Adenoviral backbone plasmid that lacks E1 and E3. pAdEasy-2 Adenoviral backbone plasmid that lacks E1, E3, and...

...assembled into a Level 2 vector, forming a functional genetic circuit. Level 2 vectors are often designed...of three sets of cloning vectors (Level 0, 1, and 2) which can be used in successive assembly steps. Before... iterations of assembly. Combining multiple Level 2 vectors permits the creation of even more complex ... 16 RBS strength variants, 8 tag-compatible RBSs, 2 secretion tags, 6 CDSs, 9 terminators, 4 nonfunctional...of multigene constructs. PLoS One . 2011 Feb 18;6(2):e16765. doi: 10.1371/journal.pone.0016765. PMID: ... Expression John Dueber 96 standardized and characterized parts that can be used for bottom-up hierarchical...constructs expressing GFP at 18 levels for rapid characterization in new organisms. Mobius Assembly Vector Toolkit...