why my plasmid became shorter? - (Jul/18/2007 )

I used pCR2.1 TOPO for TA cloning of PCR products of 200bp.
the size of PCR2.1 is 3931 bp.

after I digested the plasmid with Hind III endonucleases, I get only one band about 2kb.

What is the matter? why the plasmid become shorter?

Thank for answering.

have you tried a different restriction enzyme to reconfirm your results?

there are several possibilities for your observation
1- the plasmid was not cut with HindIII, due to insufficient digestion time, dirty DNA, mistake in making the digestion cocktail or the hindIII enzyme is dead.

2- there are 2 HindIII sites, which cleaved your plasmid into 2 fragment, both about 2kb.

3- that plasmid in your tube is not your plasmid. Just some random bit of junk

4- there is something wrong with the molecular weight ladder, maybe you made a mistake counting the bands.

5- there was something wrong with the running of the gel. Too much DNA loaded.

is it possible because the conformation of plasmid?

I just wander how to know the real size of plasmid.

by cuting, can i see the real size of plasmid by electrophoresis.

If the plasmid has been linearised by a restriction enzyme, the plasmid's tertiary conformation will be removed. It is normal practise when trying to determine the size of a plasmid, to first linearising it before running the sample on a gel.

If the plasmid has been linearised by a restriction enzyme, the plasmid's tertiary conformation will be removed. It is normal practise when trying to determine the size of a plasmid, to first linearising it before running the sample on a gel.