Reuse of Plasmid with Insert from Agrobacterium cells

[molbio123]

Here is my problem:

A year ago, I isolated a plasmid with nsert from Agrobacterium cells. Then, I used E. coli competent cells to heat shock and transform the plasmid DNA onto the agar medium I use. I picked out a few colonies, cultured them in liquid media, then isolated & purified the plasmid DNA from the E. coli cells. I ran RE digest and everything looks great. I just rechecked the DNA on another RE digest and it still looks good.

I want to keep using this stock of plasmid DNA in the future by making it up in larger batches soon. Since I isolated and purified the plasmid DNA a while ago, I will be running low on it soon. My question is, could I take this purified plasmid DNA, inject a small volume of it back into E. coli competent cells, repeat another heat shock, and retransform onto my agar medium and just grow up more this way?

I don't know of any other way to get more of this plasmid DNA and I don't want to go back to the beginning because this purified DNA looks so great. Will my way work or are there any protocols for this??

Thanks.

[admin]

Hello there and welcome to the forums Molbio123,

Ive had this problem before.

Simply take Xul of your bacteria culture (or you you can take a colony of your amazing bacteria and regrow them in selectable media such as you did).

Take Xul of glycerol and add to bacteria culture.

Carefully mix and then store in -80C freezer in aliquots.

When you need more, simply thaw an aliquot and Grow in media.

Extract plasmid using mini or midi or maxi prep kits!

Cheers!

[molbio123]

Hello,
Thanks for the reply! Yes, I am planning on freezing down a stock this time. I hadn't known I needed them so much before.

But how do I get more of this great stock first?? Could I inject a small amount into E. coli competent cells (DH5a strain), heat shock , and retransform onto agar medium? Will this produce colonies again? Thanks.