LD-PCR for MaSp

 Yesterday, I once again performed the amplification of the target MaSp gene from lyoni spider. Previously, It was difficult to amplify a large amplicon about 10kb using a set of primers that I designed. Yeah, actually it worked and the band was like 10-11kb but it was severely smeared. Since then, I tried so numerous optimization steps to obtain a good band but everything was in vail. I had spent almost 5 months just to do that and finally I gave up. After discussion with my SV, he suggested me to go fragment by fragment via primer walking. And so, I did. I redesigned all the primers and started again. At first, I performed a typical pcr condition but again no band. Then, I incorporated the phusion polymerase to amplify the band using long-distance pcr method. Interesingly, I managed to obtain a good, sharp band of 10kb amplicon that was previously smeared! Thank God!