I am doing a P element mobilization to generate a mutant of my gene. The P element is sitting 67 bp upstream of the 5'UTR of my gene. I am presently doing a pcr screen of the genomic DNA from these lines to get the desired mutant lines. I have designed a forward primer aroung 100 bp upstream of the P elemt and several reverse primers downstream at 1 kb, 2 kb, 3kb distance.
My problem is when I am doing pcr with 2 kb and 3 kb reverse, I am getting single precise band at 2 kb/3 kb distance in most of the lines (The isolated DNA was from balanced flies). I never see any line with two band which is actually expected.
To add to it, there are also few lines which do not show any amplification.
The lines are all wt eyed lines. How can I interpret these results? Please help!