IF protocol following FISH
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”
Older PCR protocol
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”
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”
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