We narrowed to 7 results for: hal.1

1. Isolating a Monoclonal Cell Population by Limiting Dilution

   Type

   Protocol

   ...Because this is such a small volume, first make 1 mL of a 1:100 dilution of the homogenized cell solution... were transduced with lentiCas9-Blast 1 and then selected with 1 μg/mL blasticidin for 9 days. Single ...with lentiCas9-Blast 1 . A549 cells were transduced (MOI = 37) and selected with 1 μg/mL blasticidin for...conditioned medium (see below for more details) Day 1: Seed individual cells in a 96-well plate Day 2–14...final 5 cell/mL solution, transfer 12.5 µL of the 1:100 dilution to 10 mL of conditioned medium. Transfer...cells will appear as colonies in the well ( Figure 1 ). You will be able to tell if there was more than...transgene expression ( Figure 2 ). Sample Data Figure 1: Generation of monoclonal cell lines from expansion...

2. AAV Titration by qPCR Using SYBR Green Technology

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   Protocol

   ...dilution Dilution 1 (DNase step) 5 uL AAV stock 45 uL 10X 10X Dilution 2 5 uL Dil. 1 95 uL 20X 200X Dilution...range from 1 x 10 12 GC/mL to >2 x 10 13 GC/mL and we use an internal reference virus that is 1 x 10 13 ...Equipment qPCR instrument Heating plate Pipettors 1–10 µL single channel pipette 20–200 µL single channel...channel pipette 200–1000 µL single channel pipette 1–10 µL multichannel pipette 2–50 µL multichannel pipette...The reference material should have a titer within 1-log of the expected titer of the samples being tested...Amount for ONE Reaction Amount for 100 reactions (1 x 96 well plate) Universal SYBR Master Mix 2X 10 μL...mole)/bp = 4.03 x 10 6 g/mole Moles/μl: 1.07 μg/μL x 1 g/10 6 μg x1 / 4.03 x 10 6 g/mole = 2.65 x 10 -13 ...

3. Gibson Assembly Protocol

   Type

   Protocol

   ...ssDNA. (Rabe & Cepko, 2020). Incubate the mix for 1 hour at 50 °C or follow manufacturer's instructions...part, one part has half of the antibiotic gene and the adjacent part has the other half. Any colonies should...

4. Virus Protocol - Generating Stable Cell Lines

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   Protocol

   ...mL polybrene (µL) 0 0 500 1:5 300 200 1:10 150 350 1:50 30 470 1:100 15 485 1:500 3 497 Add 0.5 mL of a... if an MOI >1 was used, some cells may have 1 copy of the transgene, while others have >1 copy of the ... well by pipetting or inverting the tube. Aliquot 1 mL of cell suspension (i.e., 50,000 cells) into each... early polyclonal populations. Sample Data Figure 1: Generation of monoclonal cell lines from expansion...transduced with lentiCas9-Blast and then selected with 1 µg/mL blasticidin for 9 days. Single cells were ...genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nature Methods. 2014 Aug;11(8):783-...

5. AAV Production in HEK293 Cells

   Type

   Protocol

   ...7.5% Sodium Bicarbonate, 7.5 mL 1 M HEPES to 750 mL DMEM + 1 g/L glucose. 1 mg/mL polyethylenimine (PEI) ...Calculate the amount of each plasmid needed to have a 1:1:1 molar ratio with 2 mg total DNA per CS5 Plasmid ... determine the total μg/bp we need to achieve a 1:1:1 molar ratio of each plasmid: 2000 μg / 24,961 bp...deionized water + 100 mL of 1 M Tris HCl pH 8.5 + 60 mL of 5 M Sodium Chloride + 4 mL of 1 M Magnesium Chloride...high glucose, Corning 10-013-CV DMEM, low glucose (1 g/L) glucose, sodium pyruvate, Corning 10-014-CV (... sodium bicarbonate, Corning 25-035-CI (optional) 1 M HEPES, HyClone SH30237.01 (optional) L-alanyl-L-glutamine...cations can affect the attachment of adherent cells) 1 mg/mL Polyethylenimine (PEI) 25 kDa MW Pro-Tip Other...

6. CRISPR Library Amplification

   Type

   Protocol

   ...Vented Falcon Tubes at 30-37 ℃, 225 rpm for 1 hour. After the 1 hour shaking period, pool and gently mix ...Last Update: August 17, 2023 Workflow Timeline Day 1: Transform, recover, set up overnight growth (Estimated... (Default: 4 tubes of Endura Duos, Lucigen, 60242-1) Alternatives include Stbl4 cells or other ultra-high...0.1 cm ) 20 mL SOC recovery media (Lucigen, 80026-1) 8X LB Agar + Antibiotic 245 mm bioassay plates (Molecular...into each of four 14 mL Vented Falcon Tubes and have 1 mL SOC per electroporation readily available for post-electroporation...autoclaved, sterile reagents for all steps. Procedure Day 1 Add 200 ng DNA to each 50 µL aliquot of thawed Endura...: Bio-Rad Micropulser Ec1 0.1 cm cuvette, 1.8 kV, 1 pulse. Aliquot 25 µL DNA-Endura into pre-chilled cuvette...

7. Weighing Reagents Protocol

   Type

   Protocol

   ...resuspended in a small volume (e.g. 0.02 g to resuspend in 1 mL), you may place a microcentrifuge tube on the balance...Pro-Tip If you’re using weighing paper, fold it in half first to create a crease and then place it unfolded...