After weeks of fixing frog embryos for an in situ hybridization, we eventually made RNA probes with our respective genes. The first step for in situ hybridization probes is to digest the DNA with appropriate restriction enzyme to linearize the template and extract with phenol one, add 1/10 volume of 10M NH4Oac and 2.5 volume of Ethanol, precipitated at -70 degrees Celsius for 15 minutes. Then, the DNA is spinned down in a cold room and washed with 70% Ethanol (RNA-ase free), then redissolved in DEPC-treated water, afterwards the concentration is measured. The linearized template is stored in the freezer at -20 degrees Celsius for future use. Then, we use a T7 transcription kit. The concentration of the Dact1 template is 60 nanograms per microliter, therefore, I add 4 microliters of water, 10 microliters of Dact1 template, 2 microliters of the Dig, 2 microliters of the 10X buffer, and 2 microliters of the enzyme, adding to a total of 20 microliters. This is then incubated at 37 degrees Celsius for 2 hours. Afterwards, 1 microliter of RNAse free DNAse 1 is added, then incubated at 37 degrees Celsius for 15 minutes. Then, we take 1 microliter out of the reaction and mix it with 3 microliters of RNA-gel loading dye, heated at 65 degrees Celsius for 10 minutes. This is then loaded on agarose gel. We add 15 microliters of NH4Oac stop solution and 115 microliters of DEPC-treated water to the rest of the RNA reaction. Then 150 microliters of of isopropanol is added, and precipitated at -20 degrees Celsius for 30 minutes. The RNA is spinned down and washed with RNAse free 70% Ethanol, redissolved in 20 microliters of DEPC-treated water. We take 1 microliters of the reaction to measure the concentration by the spectrophotometer. The RNA is diluted into 10 microgram per milliliter by hybridization buffer and stored at -20 degrees Celsius.
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