...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...

...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...

... 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-...

...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...

...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...