I performed sequencing after cloning those two bands. The upper band gave me full length sequence, However the lower band had some missing nucleotides. When I did the clustal allignment, I found out that 21 nucleotides from the end of exon 3 along with exon 4 & 5 is missing. Also initial 60 nucleotides from exon 6 is missing. What could be the reason of such an splicing event.???
Thank you.. But the lower band adds significance to my study. Hence I cannot just ignore it. I wanted to know how and why the band is appearing. Is it a kind of spicing event ?? Or this could be an outcome of something I am not aware of..
May be your primers are binding to all other isoforms. check the primer binding location and also other isoform of your gene. Based on your need redesign the primers.
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