Just curious to know, when carrying out a transformation can you use a plasmid that has been purified from a previous transformation, i.e. if we are running low on our plasmid, can we use the same material to transform cells again?
If not, why is there a difference between pre- and post-transformation plasmids? My only thoughts are that it might be possible that plasmid cloning per transformation introduces errors in the DNA sequence, similar reason why we don't passage reference stock cells. And to continue with IF's, if the plasmid has been confirmed to be the right sequence, is it okay to use it then?
You could transform again your bacteria if you need to renew your stock of plasmid.
I isolate one clone on LB agar, do mini and midi culture. In addition, I keep a "glycerol stock" of the bacteria to keep live cells with the plasmid.
Because you start with one bacteria with one plasmid, all cells keep the same plasmid. The replication of plasmid is not mutagen. But yes you could sequence your gene to be sure.
Replication systems are not 100% faithful, so there will always be a small proportion of plasmid molecules in every preparation that will bear unwanted mutations. Sequential transformation/purification cycles increase the chances that you will pick up a mutated version of the original plasmid (especially in hosts defective in repair systems, such as dam- strains), so it is good practice to stick to the original prep if at all possible -although in real life, you'll be hard-pressed to find cases where this has actually posed a problem.
Of course, that point is moot if you have time/resources to sequence the new prep.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques