Sharpening the Big Mama MaSp-CL

Since the day I obtained the >10kb band of MaSp-CL, I have been working out so hard to sharpen the band by optimizing the PCR condition. Nonetheless, I still could not get the sharp, intense band as desired. Previously when I tried with gDNA as template, I face the smearing problem though I have catered the template concentration problem. It turned out that the primer design was not proper thus, after designing a new primer set, band of more than 10kb emerged but alas, it was not the sequence that I was looking for due to the non-targeted primer design@cross-species primer. However, this time I designed a targeted primer based on the MaSp-CL putative sequences from the transcriptomic analysis. Though I had obtained the band of desired, I just could not make it sharp and intense..But, I will try very hard tomorrow again with new cDNA samples! Crossing fingers to this!

 

So disappointing when getting this type of result...