... confluent, expand into larger vessels. A confluent well of a 6-well dish can be expanded into a 10 cm...reagent Day 3–14: Change media as needed Day 14–18: Expand and harvest stable cell lines Equipment Class II...undergo additional selection while the population expands. At this time, some researchers reduce the concentration...cm dish. A confluent 10 cm dish can be expanded into two 75 cm 2 flasks, etc. Pro-Tip This selection method...populations are growing well and have been sufficiently expanded, prepare cell stocks and/or harvest to test for...will have higher levels of expression. Consider expanding populations transduced with a variety of dilutions...Figure 1: Generation of monoclonal cell lines from expansion of individual A549 cells stably expressing Cas9...

... Monitor cells for growth and expand cells Day 14–30: Analyze and expand monoclonal lines of interest ...cells have expanded but before they become over-confluent, trypsinize the cells and expand them to larger...After the monoclonal lines have been sufficiently expanded, screen the lines for transgene expression and...Figure 1: Generation of monoclonal cell lines from expansion of individual A549 cells stably expressing Cas9... in individual wells of a 96-well plate after expansion of single cells for 13 days. Note that growth ...Single cells from a stable cell pool were then expanded for an additional 3 weeks under blasticidin selection...stable cell pool (pool) and from individual cell expansions (monoclonal, 1-5). Wild-type A549 cells do not...

...). You can use either Isopropanol or Lithium Chloride for this step. Isopropanol (Option A): Add 1 volume...). You can use either Isopropanol or lithium Chloride for this step. Isopropanol (Option A): Add 1 volume...precipitation step, two options are also included: using Isopropanol or Lithium Chloride. This protocol was adapted...decontamination solution like RNase AWAY® or RNaseZAP® Isopropanol (for precipitation step, Option A) 7.5 M Lithium...volume of Isopropanol to the extracted aqueous layer. Incubate at -20°C for 1 hour. Lithium Chloride (Option...

...bacterial cultures , please see those pages. Several companies, such as (Link opens in a new window) Qiagen ,...phenol-chloroform Chloroform 100% ethanol or isopropanol 90% ethanol 70% ethanol TE buffer 3 M Na-acetate...plasmid-containing supernatant. Add either ethanol or isopropanol to precipitate the plasmid DNA. Either spin to...for 5 min. Note: Ribonuclease A (RNase A) is a pancreatic ribonuclease that digests single-stranded RNA...cold 100% ethanol or 350 μL room temperature isopropanol to the solution to precipitate the plasmid DNA...

...containing the trypan blue. Pipette 10 times to mix. Load 10 µL of the cell suspension/trypan blue mix into....45 µm PES filtering system, 500 mL, VWR 430770 Trypan Blue, Thermo Fisher T10282 Valproic acid sodium...flask 5–10 times before sampling. Transfer 10 µL of trypan blue into a clean microcentrifuge tube. Vortex ...

...limited MCS (BamHI - EcoRI - SalI) and you want to expand that with the addition of NdeI, PacI, AscI, and...following oligos from your favorite oligo synthesis company: Top oligo: 5' - AATTCCATATGTTAATTAAGGCGCGCCCAATTGG...

...positions he has held in his career, his favorite company tradition, and Addgene's role in the scientific... offers career advice for scientists looking to expand their international reach. Joel McDade Joel McDade...

...commonly referred to as 'competent cells.' Many companies sell competent cells, which come frozen and are... You should also add a positive control (many companies include a positive control plasmid with their ...

...without limiting cell growth. Left panels: bright field images; right panels: GFP channel images. Last reviewed...

...985 (Link opens in a new window) . Figure 2: Left panel: Iodixanol gradient after ultracentrifugation. The...): 973-985 (Link opens in a new window) . Right panel: cartoon indicating the position of the needle for...

...until no live cells can be seen when stained with Trypan Blue. Pellet cell debris by centrifugation at 3900...started with cells that are ~80% confluent (center panel). These cells were imaged with a 10X objective and...

...don’t touch your face! Close-toed shoes and long pants should be worn to protect your legs and feet from...

...trace file (.ab1 file) provided by your sequencing company or core facility The corresponding primer sequence...

... with microfuge tubes in place, and the control panel where you can set the time and speed needed for ...

...buffer that works for both of your enzymes. Most companies will have a compatibility chart, such as the (...

...fluorescent protein expression (green in the right panels) with a limited effect on cell growth. Last reviewed...

...Diluting Primers Most people order primers from a company, which synthesizes and ships them as a lyophilized...

...electroporation. Arcing would manifest as a loud pop often accompanied by some light in the cuvette during the electroporation...