Plasmid sequencing - (Apr/04/2005 )
I got my clones by restriction enzyme digestion checking. I did midi prep to get it sequenced. what primer i should use to sequence it? i get confused about sequencing, do you get all plasmid sequenced or only sequence the part of insert. should i design the primer for sequecing?
my insert are different sizes, 200bp, 1.6kb. one of my collegue said i should design a special primer in the middle of 1.6kb insert to get it fully sequenced, true or not?
It depends on the plasmid you are using. Usually sequencing facilities have standard primers for most plasmids and you don't need to design or even provide such primers for sequencing. Just tell them what plasmid it is, they will know what primer to use. Those standard primers are usually located at both sides of multiple cloning site (MCS). You can use either the forward or reverse primer. If you need accuracy, sequence using both primers. For your 1.6 kb insert, you may need to design 1-2 primers in the middle of the insert, although the whole insert can be covered by sequencing from both directions without an internal primer.
You only need to sequence the insert.