On-column removal assay for DNA contamination

Summary

This protocol can be used for RNA isolation strategies using silica-based RNA binding columns, such as protocol 6, however, 100% DNA removal may not be achieved. this experiment was derived from PCR Lab Guide (Second Edition) by Kang Seed and Lijia Qu.

Operation method

On-column removal assay for DNA contamination

Materials and Instruments

DNA Enzyme I Buffer DNA Enzyme I
Cell lysate

Move

I. Materials

1. Enzyme and enzyme buffer

DNAase I, amplification grade (no RNAase)

DNA Enzyme I Buffer, 10X

2. Cells and tissues

Cell lysates obtained using a total RNA purification kit (e.g. Madigen Total RNA Rapid Purification System).

II. Methods

1. Apply the lysate to the centrifugal sleeve column included in the Total RNA Purification Kit (e.g., Marligen Total RNA Rapid Purification System).

2. After loading the lysate onto the RNA Purification Membrane, add 50ul of DNAzyme I solution containing 5UD DNAzyme I in 1X DNAzyme I Buffer to each centrifuge cartridge.

3. incubate at room temperature for 15 min.

4. Continue the Marligen purification protocol at step 6 in Scheme 6, Method II. The washing step later in the protocol will discard the DNAase I.

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