SOUTHERN BLOT ANALYSIS FOR 21-MER IDENTIFICATION FROM pDR3.3

摘要

Southern transfer and hybridisation was used to check the presence of a particular DNA sequence in aplasmidial DNA sample. In this procedure, the plasmidial DNA(pDR3.3) was isolated from E. coli strain (HB1O1::TnS) cells and then digested with EcoRI and ClaI /XhoI restriction enzymes. The DNA restriction fragments were then loaded onto a 1 % agrose/TAE gel and the fragments were separated by electrophoresis according to size, with the smaller fragments migrating faster than larger fragments. The DNA was then transferred from the fragile gel to a nylon filter which has an improved capacity for DNA binding. The DNA was treated with alkali by sequential soaking of the gel in solutions containing NaCl and NaOH, respectively. This denatures the double - stranded DNA to produce single strands. Following transfer, the DNA was fixed to the membrane by illumination with UV irradiation. Next the 3 'End-Labelling of probe with biotin was added. The probe DNA was hybridised to the complementary single-stranded DNA fragments on the filter paper. To detect the position of the radioactive probe the nylon membrane was exposed to X-ray film and the resulting autoradiograph showed the positions of the complementary fragments of DNA.