Clinical isolate of S.aureus showing resistance to ampicillin was chosen in the present study for isolation and amplification of ß-lactamase gene was successfully amplified from chromosomal DNA using the primers constructed from class (A) ß-lactamase gene of M.tuberculosis H37Rv. PCR product was cloned into the SmaI site of M13 mp18 vector and positive clone was selected using 100µg/ml ampicillin and further, the DNA from the clone was isolated and checked for the insert using the same primers. PCR product (0.7Kb) indicated that the cloning was successful which also confirmed that ß-lactamase that confers resistance to ampicillin and to other ß-lactam antibiotics was present on the chromosomal DNA of S.aurues.