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 22X30 coverslip, seal…

Older PCR protocol

You should first run qPCR to adjust template concentration (see real-time PCR). 4X linear PCR: ReagentInitialFinal1X reaction (50 μl)4.5X (μl)Kapa Buffer A10x1x522.5Forward10 µM0.4 µM29Reverse (T7)10 µM0.4 µM29dNTP mix10 mM0.4 mM29Raw library2 ng/µL0.2 ng/µL522.5Water 33.5150.75Kapa Taq5 U/µL0.5 U/µL0.52.254X linear PCR

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 when…

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 5…


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