Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

Help! my plasmid size changed after ligation - (Aug/16/2010 )

My purpose is to clone a fragment(500bp) in to a 7500bp vector. I used KpnI and XhoI to cut both the vector and the insert. (KpnI or XhoI cuts the vector only once)
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...

-misakid-

Did you check whether your insert has a KpnI-site too, approximately on the opposite site of your vector-KpnI-site, thereby cutting your plasmid into two fragments of nearly similar length

-ElHo-

What is the size of the plasmid that originally contained your insert? Set up the ligation with the proper control (ie. insert with no vector) and you will probably find colonies. Screening ligations by pcr is not the best choice if using insert specific primers.

-NemomeN007-

If you use colony PCR to screen, always PCR amplify the junction between insert and vector. A PCR for the insert will very likely yield a false positive. PCR is powerful enough to detect DNA fragments (left over from the ligation reaction) sitting on the plate. Only the junction between insert and vector is unique.

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.

-perneseblue-

Thank you all. KpnI only cuts the plasmid once. Later I checked more colonies. Some colonies have plasmid of correct size, while some have the wrong size. Anyway I'm using the correct coloniesto check sequence.

-misakid-

misakid on Mon Aug 23 04:56:16 2010 said:


Later I checked more colonies. Some colonies have plasmid of correct size, while some have the wrong size.


I see this all the time...

-HomeBrew-