Help! my plasmid size changed after ligation
Posted 16 August 2010 - 10:29 PM
After RE digest, I ran a gel and extracted the vector, which is about 7500 bps.
After ligation and transformation, I extracted plasmid and did PCR using primers for my insert. The gel picture shows that my insert is there.
But when I use KpnI to digest, the gel picture shows that the plasmid becomes about 3500 bps.
I really cannot understand why...
Posted 17 August 2010 - 03:56 AM
Posted 17 August 2010 - 04:52 AM
Posted 17 August 2010 - 09:24 PM
As, for the KpnI/XhoI digest, do you see the vector backbone? Is that the right size?
How many colonies were checked? It is possible that this one colony is the wrong thing.
Posted 22 August 2010 - 08:56 PM
Posted 23 August 2010 - 01:41 AM
Later I checked more colonies. Some colonies have plasmid of correct size, while some have the wrong size.
I see this all the time...