Day-of-experiment protocol, including fluidics

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”

FISH with #1.5, 40 mm coverslip

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”

Protocol for Tetraspeck Calibration Slide Preparation used for generating Biplane PSF

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”

Real-time PCR

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”

FISH on ibidi channel slides

Updated: 03/31/2022 For super-resolution, you can choose to either glue your choice of coverslips (preferably 1.5H) to a sticky channel slide or use the premade channel slide VI 0.5. For fluidics applications use the u-slide I Luer. Constructing the channel (if using sticky-slides): Sonicate coverslips 10 minutes in 1:11 Branson solution, then rinse thoroughly, sonicateContinue reading “FISH on ibidi channel slides”

Cleaning MatTeks

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”

Probe amplification using T7

Updated 08/08/22 Linear PCR: pre-amplification of the entire library Twist recommends 0.4 ng/µL template concentration. 2X linear PCR: Reagent Initial Final 1X reaction (50 μl) 2X (μl) Roche Diagnostics KAPA HiFi HotStart ReadyMix PCR Kit (6.25 mL), 250 x 50 uL reactions 2x 1x 25 50 Forward 10 µM 0.3 µM 1.5 3 Reverse (with T7 promoter) 10 µMContinue reading “Probe amplification using T7”