Vortex the tube vigorously for 15 seconds until completely resuspended. Then discard as much supernatant as possible without disturbing the visible white pellet. Centrifuge at 600g for 10 minutes at room temperature. Invert the tube several times during a 10-minute incubation period at room temperature. Then transfer blood to a sterile 15-milliliter centrifuge tube containing nine milliliters of cell lysis solution. For DNA isolation from blood, gently rock the tube until it is thoroughly mixed. After the incubation, allow the sample to cool to room temperature for five minutes. Then place a biopsy into the tube, and incubate it at 65 degrees Celsius for 15 to 30 minutes.Īdd 17.5 microliters of 20-milligrams-per-milliliter proteinase K, and incubate the tube overnight at 55 degrees Celsius, with gentle shaking. Add 600 microliters of nuclei lysis solution to a sterile 1.7-milliliter microcentrifuge tube, chilled on ice. If using frozen biopsies, thaw them on ice. Retrieve intestinal biopsies either freshly collected or stored at negative 20 or negative 80 degrees Celsius. Begin by performing digestion and cell lysis of intestinal biopsies. This has the potential for identification of novel biomarkers in diverse diseases.ĭemonstrating that procedure will be Naomi Salamon, a research assistant from my laboratory. The advantage of next-generation sequencing is the ability to identify unique T or B cell clones and track them in different anatomical sites or in different individuals.
This method enables exploration of specific T and B cells at the nucleotide or amino acid level and thus can identify dynamic changes in lymphocyte populations and diversity parameters in different disorders.
0 kommentar(er)
