Sealy Center for Structural Biology and Molecular Biophysics

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 22X30.

Last updated: 02/08/19 C01beta NonHOPs library: Reagent Initial Final 1 reaction (μl) Kapa Buffer A 10x 1x 10 Forward 10 uM 0.4 uM 4 Reverse (T7) 10 uM 0.4 uM 4 dNTP mix 10 mM 0.4 mM 4 Kapa Taq 1 Raw library 0.2 ng/ul 1µl per 10µl of reaction 10 Water (up to 100 ul) 67 C01beta HOPs library and HOPs1 of HOPs_12_49.

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 the

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.

Last updated: 11/13/19 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.

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.

Posted 05/23/2016 FISH with ibidi sticky-slide VI 0.4. Adapted from Brian and Hiroshi; Here’s a movie on IF and µ-Slide VI for your reference; Application note for IF and µ-Slide VI for your reference; FISH steps were adapted from Brian Beliveau and Hiroshi Sasaki from Peng Yin’s laboratory.

Updated 09/30/20 Linear PCR: pre-amplification of the entire library (protocol adapted from Mo and Sonny) You should first run qPCR to adjust template concentration (see real-time PCR page)...

Posted 09/22/2015 For STORM I’m using 35 mm MatTek dishes. Their surface area is ~8 times smaller than 100 mm dish. If I’m splitting IMR90 1:5, then I’m taking 1 volume of that and diluting 1:8, in total, a dilution of 1:40 and then seed 8 35 mm dishes, 2 ml each...