Investigation of the factors that influence long range PCR / Muhamad Saifuzzaman Mohamad Kassim

Long range PCR method for uniform amplification of specific lambda DNA sequence is described. The careful design of all 6 pairs of primers and optimum temperature cycling profile produce good result for long range PCR. Three pairs of 15kb and three pairs of 20kb primers were design using oligo explorer 1.2. Four out of six (All three 15kb PCR and one 20kb PCR) produce results. Two variables that affect the efficiencies of long range PCR were studied which consists of the percentage of DMSO added and the concentration of MgCb used in the PCR. A combination of 5%, 7%, and I 0% DMSO were added to study the improvement of the efficiencies of long range PCR amplification that theoretically should increase the ratio of full-length products to shortened products. Results were somehow unobtainable and optimum percentage of DMSO for long range PCR is not known. The differing MgCb concentrations were added and the result was also unobtainable and optimum concentration of free Mg2+ in the PCR mix is not known.