Skip to main content
Addgene

Enter a sequence to perform a BLAST-based search of Addgene plasmid sequence databases.

In Category

We narrowed to 492 results for: ara-2

Showing: 481 - 492 of 492 results

  1. Transfection for Recombinant Antibodies

    Type
    Protocol
    ...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...

  2. Lentivirus Production

    Type
    Protocol
    ... 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...

  3. Immunocytochemistry

    Type
    Protocol
    ...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...

  4. 9 tips for a successful postdoctoral experience

    Type
    Blog Post
    ...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 ...

  5. Ligation Independent Cloning

    Type
    Protocol
    ...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...

  6. Virus Protocol - Generating Stable Cell Lines

    Type
    Protocol
    ...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 (...

  7. CRISPR Library Amplification

    Type
    Protocol
    ...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...

  8. Protocol - How to Inoculate a Bacterial Culture

    Type
    Protocol
    ...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... to do a mini-prep you will usually want to start 2 mL in a falcon tube, but for larger preps you might...might want to use as much as a liter of LB in a 2 L Erlenmeyer flask. Using a sterile pipette tip or toothpick...After incubation, check for growth, which is characterized by a cloudy haze in the media (see right). Notes...

  9. Protocol - Over-Agar Antibiotic Plating

    Type
    Protocol
    ... 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...

  10. Isolating a Monoclonal Cell Population by Limiting Dilution

    Type
    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-...

  12. Plasmid Cloning by PCR (with Protocols)

    Type
    Protocol
    ...DNA concentration alone. One method is to conduct 2 ligations for each plasmid you are trying to create... also important to set up negative controls in parallel. For instance, a ligation of the recipient plasmid...
Showing: 481 - 492 of 492 results