We narrowed to 5 results for: hal.2

1. 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...medium at a reduced antibiotic concentration (e.g., half the concentration that was used for the initial ...

2. AAV Titration by qPCR Using SYBR Green Technology

   Type

   Protocol

   ... 10 90 2 x 10 8 10 of 2 x 10 8 dilution 90 2 x 10 7 10 of 2 x 10 7 dilution 90 2 x 10 6 10 of 2 x 10 6...6 dilution 90 2 x 10 5 10 of 2 x 10 5 dilution 90 2 x 10 4 10 of 2 x 10 4 dilution 90 2 x 10 3 Pro-Tip...molecules/μL To obtain a solution at 2 x 10 9 molecules/μL: 1.59 x 10 11 / 2 x 10 9 = 79.8X dilution ...your standard curve plasmid (2 x 10 9 stock made in step #1): Volume of 2 x 10 9 stock or previous dilution...stock 45 uL 10X 10X Dilution 2 5 uL Dil. 1 95 uL 20X 200X Dilution 3 20 uL Dil. 2 80 uL 5X 1000X Dilution ...time required: ~3 h Workflow Timeline Plate set-up: 2 h qPCR run: 1.5 h Data analysis: 30 min Equipment ...single channel pipette 1–10 µL multichannel pipette 2–50 µL multichannel pipette 20–200 µL multichannel ...

3. AAV Production in HEK293 Cells

   Type

   Protocol

   ...430825, 175 cm 2 Cellstack 5, Corning 3319, 3180 cm 2 Cellstack 2, Corning 3269, 1272 cm 2 Heat-inactivated...: 50 mM Tris HCl, 150 mM NaCl, 2 mM MgCl 2 Add the following to the 2 L sterile bottle: 1836 mL deionized... solution can be stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working... mL of PBS. Aspirate PBS and add 2 mL of 0.05% Trypsin/EDTA. Wait ~2 min. Neutralize trypsin by adding...Cell-Stack (CS5) (Link opens in a new window) (3,180 cm 2 - the same surface area as 21 x T-175 flasks). Cell...flasks. Workflow Timeline Day 0: Seed cells in CS2 Day 2: Seed cells in CS5 Day 3 (am): Transfect cells Day...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...

4. 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...genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nature Methods. 2014 Aug;11(8):783-...

5. CRISPR Library Amplification

   Type

   Protocol

   ... 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...Quality Control (QC) Pooled libraries can be challenging and expensive to ensure adequate quality, but...