I digest my plasmid vector by using 2 different restriction enzymes. My PCR product is a ligated product of 4 different genes (gene 1 to 4) . My PCR product also having matching sites of vector at its both ends (gene 1 and gene 4). After cloning and colony PCR I found that band size of my clone is less than that I am expecting. After getting sequencing results I found that gene 1 sequence was missing and rest three gene sequences are present. As gene 1 has a matching sequence for vector, I would like to know how it is possible to get clone without having gene 1 sequence? I checked sequencing results by using NCBI Blast.
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