RNA purification using TRizol TRIZOL法提取RNA【University of Florida】

a. Take 2 to 5 μl RNA sample from the original stock, diluted with 998 or 995 μl RNase free water in a 1.5 ml microcentrifuge tube. This will give you 500 or 200 time dilution of the RNA sample.

b. Pipet 1 ml RNAse free water in a clean cuvette and read absorbance as blank.

c. Pipet the diluted RNA sample in to a clean cuvette and read absorbance at 260 nm and 280 nm.

d. Use the formula below to determine RNA Concentration of the original sample:

[RNA μg/μl]= A₂₆₀ x 33 x dilution factor / 1000

e. To determine the purity of the RNA sample, calculate ratio of A260/A280. Ratios between 1.7 to 2

represent good RNA)

Preparation of RNase-free water

a. Measure water into RNase-free glass bottles.

b. Add 0.01% (v/v) diethylpyrocarbonate (DEPC).

c. Let stand overnight.

d. Autoclave.

Note: RNase free DEPC treated water is Biotecx brand (cat # BL-5611).

Note: If using 0.5% SDS solution to resuspend the RNA. It must be prepared in RNase free water.