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-   -   Reuse of Plasmid with Insert from Agrobacterium cells (http://www.molecularstation.com/forum/dna-techniques/1855-reuse-plasmid-insert-agrobacterium-cells.html)

molbio123 08-04-2007 06:05 PM

Reuse of Plasmid with Insert from Agrobacterium cells
 
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 08-05-2007 05:22 AM

Re: Reuse of Plasmid with Insert from Agrobacterium cells
 
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 08-05-2007 05:47 PM

Re: Reuse of Plasmid with Insert from Agrobacterium cells
 
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.


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