doubt regarding agrobacterium DNA restriction digestion

Michael Sullivan via methods%40net.bio.net (by mlsulliv from wisc.edu)
Wed Jun 18 12:16:25 EST 2008


Are you trying to digest plasmid DNA from agro to make sure you have  
transformed a binary vector construct?

Yield and quality of DNA from agro are usually much lower than you  
would get from E. coli, so sometimes it is difficult to directly  
analyze DNA isolated from agro.

I have had good luck using Qiagen plasmid minipreps. Usually, I get  
DNA of good enough yield and quality to see restriction digestion  
products. In the past, using simple alkaline lysis type procedures,  
results were typically more variable- sometimes I could see digestion  
products, sometimes not. However, I found the DNA was always of  
sufficient quality to retransform into E. coli. This means that to  
analyze a plasmid from agro, you may need to transform the DNA back  
into E. coli, where prepping and doing a digest will be trivial. If  
results of the DNA analysis from E. coli look good, you know your  
plasmid in agro was OK.

Mike

On Jun 18, 2008, at 11:02 AM, priyadarsini Tripuraneni wrote:

> hi all
> as i am attempting to digest agrobacterium DNA i am not able to do  
> that.
> please help me regarding this.
>
> -- 
> prasanthi
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