Multiple bands after amping insert - (Mar/04/2010 )
My object is to sequence cloned products. I have cloned 500 bp insert in a 3 kb plasmid and after extracting plasmid, to confirm,I set up a PCR using forward primer of my vector and reverse primer of my insert. The gel image shows me multiple bands (high mol weight )in addition to expected size band. Also, few of my plasmid amps didn't have the expected size band but just high mol wt bands.
I understand that different sizes means different conformation of plasmids (linear,circular and supercoiled due to nick formation etc)? My question is my insert there in the plasmid? should I go ahead with the sequencing?
If the expected product comes up, then, yes the insert is in the plasmid. The bigger bands just mean that you have enough template plasmid in your PCR to be able to see the plasmid on the gel.
please make sure your products are not contaminated...