We describe a protocol for the insertion of ultrashort single-walled carbon nanotubes (SWCNTCNTCNTs) to form nanopores in a Montal-Mueller lipid bilayer. The SWCNTCNTCNTs are designed to bind to a specific analyte of interest; binding will result in the reduction of current in single-channel recording experiments. The first stage of the PROCEPROCEPROCEPROCEPROCEDUREUREURE is to cut and separate the SWCNTCNTCNTs. We cut long, purified SWCNTCNTCNTs with sonication in concentrated sulfuric acid/nitric acid (3/1). Isolation of ultrashort SWCNTCNTCNTs is carried out by size-exclusion HPLCPLCPLC separation. The second stage is to insert these short SWCNTCNTCNTs into the lipid bilayer. This step requires a microinjection probe made from a glass capillary. The setup for protein nanopore research can be adopted for the single-channel recording experiments without any special treatment. The obtained current traces are of very high quality, showing stable baselines and little background noise. Example procedures are shown for investigating ion transport and DNANA translocation through these SWCNTCNTCNT nanopores. This nanopore has potential applications in molecular sensing, nanopore DNANA sequencing and early disease diagnosis. For example, we have selectively detected modified 5-hydroxymethylcytosine in single-stranded DNANA (ssDNANA), which may have implications in screening specific genomic DNANA sequences. The protocol takes similar to 15 d, including SWCNTCNTCNT purification, cutting and separation, as well as the formation of SWCNTCNTCNT nanopores for DNANA analyses.