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iPOND, Part 1: Fishing for Proteins with DNA as Bait
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Steve Jobs Had LSD. We Have the iPhone TIME.com
WebHigh quality leadership abilities make Ipond into a good teacher. Pay attention to the subtle difference between teaching and instructing, so that people continue to listen to you! If … Timing 1 h 1. 38 Prepare the lysis buffer by adding aprotinin and leupeptin before use (see REAGENT SETUP) and place on ice. 2. 39 Resuspend the samples from Step 37 at a concentration of 1.5 × 107 cells per 100 μl of lysis buffer and transfer them to 1.5-ml centrifuge tubes on ice. To examine DNA fragment size at … See more Timing 1–7 d 1. 1 Calculate the number of dishes of cells needed for the experiment. Each sample requires at least 1.0 × 108cells at the time of … See more Timing 10 min–8 h 1. 3 Determine the cell number in the extra dish of cells from Step 2. This cell number will be used to calculate the amount of the reagents used for each sample in … See more Timing 1 h 1. 19 Resuspend the cells in permeabilization buffer at a concentration of 1 × 107cells per ml. 2. 20 Incubate the cells at RT for 30 min. During incubation, thaw and prepare the … See more Timing 1 h 1. 11 After EdU pulse and/or chase, decant the medium. 2. 12 Immediately fix the cells on a dish by adding 10 ml of 1% (wt/vol) formaldehyde in PBS and incubating … See more WebIsolation of protein on nascent DNA (iPOND) is an elegant method for purifying replication fork proteins. Here, we present accelerated native iPOND (aniPOND), a simplification of … inblock-shroud