1 reply to this topic

[Benny85]

I have bacteria stocks/clones that contain my plasmid of interest. The clones were previously tested to contain the plasmid of interest by restriction enzyme digestion.

Now that i need to use the plasmids again. So I inoculated the bacteria stocks onto my ampicilin bacteria media (bacteria carry this plasmid of interest with ampicilin resistance gene) and let the bacteria propagate, then did a maxi prep to obtain the plasmid.

Why is there a need to perform restriction enzyme (RE) digestion again on the plasmid obtained (which has already been tested)?

Is there something to do with the bacteria losing the plasmid during propagation? how? are there any other reasons to perform RE again?

Thanks.

Benny85

[Trof]

Bacteria can do all sort's of things with a plasmid. They can loose it, they can recombine it. Also you used a different prep, isolation, different conditions. I would check it.

Edited by Trof, 16 March 2012 - 08:35 AM.