I am trying to analyze a plasmid insertion sequence. I have the sequence and have found some motif sites of interest in the correct place, but I cannot manage to figure out the orientation of my insert in my plasmid.
When I sent the plasmid off for sequencing, I sent it with a primer, but when I search for the primer in the sequence, it does not show up. The primers that I used to create the insert also do not show up.
Any ideas on why my insert sequence does not contain the primer used to do the sequencing?
In sanger sequencing you never get sequence under the sequencing primer or for the next 20-30 bases. You will only see forward primer by sequencing back from the reverse primer . To see your entire sequence you will have to use plasmid sequence primers about 50 bases away from your insert
In Sanger sequencing we do not get sequence of first 30 bases downstream of primers. if you want to sequence ends of your insert either use plasmid sequencing primers or internal primers.
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