how to store plasmid samples? - (Aug/09/2007 )

Hi everyone...

i have a question on how should i store plasmids correctly in order to minimize mutations or contamination?

Because, this was what we did:
1. we have original plasmids stored in fridge and we did gel electrophoresis on these original plasmids
2. we transformed these plasmids into Ecoli, then extracted plasmid, finally we ran gel electrophoresis on these extracted plasmids.

Now, when we compared gel results of the original samples with the ones recovered from Ecoli, the plasmids have diferent sizes!! [the recovered plasmids had smaller sizes...]

could it be because of storage problems? what else could have caused this unbelievable result?

Your plasmid freshly isolated from E. coli might be coiled or supercoiled, and it causes the plasmid to run faster on an agarose (i.e appears smaller).

My suggestion is to a complete cut of each samples with a restriction enzyme that has a unique site on the plasmid and analyse the digest with an agarose gel.

If both cut plasmid have the same size, you're fine and if one plasmid runs faster, you got a problem.

we routinely store plasmid preps at -20°.
For every new cloning (that is interesting of course) we do midiprep and sotr 1ml of culture in glycerol and an aliquot of plasmid prep both at -80° in a lab collection resource.

In addition to purified plasmid, I also store transformed cultures at -80.