Home › Microbiology › Electricity-Free, Sequential Nucleic Acid and Protein Isolation
Microbiology JoVE (Open Access) Citable · DOI

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

DOI: 10.3791/4202-v
What you'll learn
  • ✓ Perform sequential nucleic acid and protein isolation without electrical equipment
  • ✓ Apply solid-phase extraction principles using a sorbent-filled transfer pipette
  • ✓ Prepare macromolecules suitable for PCR-based and immuno-based downstream assays
Protocol

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

Difficulty
intermediate
Total time
~30-45 min per sample
Biosafety
BSL-1

Steps

1
Lyse sample using chemistry-based lysis buffer

Add lysis buffer to the sample to break open cells and release cellular contents. This prepares the sample for sequential macromolecule extraction.

▶ 02:20
2
Extract nucleic acids via solid-phase sorbent

Use the sorbent-containing transfer pipette to bind and isolate nucleic acids from the lysed sample through solid-phase extraction principles. Elute the purified nucleic acids.

▶ 03:20
3
Extract proteins from residual sample material

Apply protein extraction chemistry to the remaining sample material after nucleic acid removal. Isolate and elute proteins suitable for downstream immunoassays.

▶ 05:27
4
Verify isolation and analyze macromolecules

Perform PCR-based and immuno-based assays on the isolated nucleic acids and proteins to confirm successful sequential extraction and assess sample quality.

▶ 07:11
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