Cloning of 6.5 kb Segment of Cell Culture Adapted Classical Swine Fever Virus

In this study, a 6.5 Kb fragment of cell culture adapted Lapinized Classical Swine Fever Virus (CSFV) was cloned into pTZ57R/T vector using the TA molecular cloning technique. Briefly, viral RNA was isolated using a modified RNA isolation protocol using Ribozol and RNeasy followed by cDNA synthesis using Clontech superscript RT. The cDNA obtained was used to synthesize 6.5 kb PCR products using (Long acting) LA-PCR. Gel purified PCR product was cloned with pTZ57R/T vector which was confirmed by RE digestion and partial sequencing. The same protocol can be utilized for cloning of similar sized amplified products in molecular biology laboratories.