Author Archives: guynir1

Last updated: 06/06/19 For FISH protocol see: FISH with #1.5, 40 mm coverslip Three washes with PBT for 10 minutes each. Wash with 3% BSA-PBT 1 hour [1.5g BSA in 50ml PBT; 1% is equal to 1 g in 100 mL]. Add 35 µl primary Ab in 1% BSA-PBT to Bioptechs coverslip and cover with 22X30Continue reading “IF protocol following FISH”

You should first run qPCR to adjust template concentration (see real-time PCR). 4X linear PCR: Reagent Initial Final 1X reaction (50 μl) 4.5X (μl) Kapa Buffer A 10x 1x 5 22.5 Forward 10 µM 0.4 µM 2 9 Reverse (T7) 10 µM 0.4 µM 2 9 dNTP mix 10 mM 0.4 mM 2 9 Raw library 2 ng/µL 0.2 ng/µLContinue reading “Older PCR protocol”

Updated: 10/01/20 Following non-bound primary oligos wash (see FISH protocol): Cleaning the fluidics before use (can be done a day ahead if the scope is available): Flush water –> ~8% RBS –> water with 600 μL in reservoirs 1-15 and 3 mL in reservoirs A-D. Rate can be set to 600 μL/min. Clean again whenContinue reading “Day-of-experiment protocol, including fluidics”

Last updated: 07/27/21  Sonicating: Sonicate coverslips: 10’ in 1:12 Branson solution, 10’ de-ionized water, 10’ Ethanol, and dry, or leave immersed in EtOH. Air-dry in the hood before placing in a tissue-culture dish. Seeding: Place 7 40 mm coverslips in a big petri dish and cover with ~20 ml media. Trypsinize cells and resuspend in 5Continue reading “FISH with #1.5, 40 mm coverslip”

Updated: 11/21/19 Note – I now add the tetraspeck beads onto my sample. That way the calibration is performed in the same environment, stage, chamber holder, and coverslip as the sample. Therefore, PSF generated in the calibration will resemble the one registered in the actual imaging. For details on how to add the beads followContinue reading “Protocol for Tetraspeck Calibration Slide Preparation used for generating Biplane PSF”

Updated 09/26/20 Follow these tables to prepare two 3.5X reactions, where you will test two different primer concentrations, each with three different template concentrations. This is done for optimizing the primer and template concentrations and their ratio. Don’t add the library (template) to the mix tube.For libraries bought from Twist use 2 pg/uL as theContinue reading “Real-time PCR”

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It is recommended to clean the MatTek bottom, especially for super-resolution/single-molecule imaging. I use BRANSON  ultrasonic cleaner 1800. make 1:12 detergent solution from the BRANSON cleaning solution (or Alconox, etc). I add 25 ml 1:12 cleaning solution to 275 ml deionized water. Degas for 5 minutes. Place MatTeks (only the bottom is enough) inside a beakerContinue reading “Cleaning MatTeks”