The following protocol is a modification of the Dilworth and Huang protocol (DilworthandMcCarey1992;Huangetal.2000). It can be used to remove DNA contamination from RNA prepared with most commercially available kits and reagents. This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.

Operation method

DNA contamination removal assay after elution

Materials and Instruments

EDTA DNAase I DNAase I Buffer RNA Sample
Water bath

Move

I. Materials

1. Buffers, solutions and reagents

Water treated with diethyl pyrocarbonate (DEPC)

EDTA, 25 mmol/L

2. Enzyme and enzyme buffer

DNAase I, amplification grade (without RNAase)

DNAase I buffer, 10X

3. Nucleic acids and oligonucleotides

RNA Sample

4. Specialized equipment

Water bath, preset at 65°C

II. Methods

① Add the following components to a tube without RNAase.

RNA not more than 80ug

DNA Enzyme I Buffer, 10X 1ul

DNAase I (lU/ul) 1ul

Bring the total volume to 10ul with DEPC-treated water.

② Incubate for 15 min at room temperature.

(iii) Add 1ul of 25 mmol/L EDTA solution and heat at 65°C for 10 min to inactivate DNAzyme I.

Samples processed in this way are suitable for direct use in RT-RCR.

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