request for agrobacterium mini prep protocol,not kit - (Jul/16/2008 )

Hi,
This is reddy,i am working on agrobacterium transformation.i transfered plasmid to agrobacterium and plated i got positive colonies,i want to make confirmation by restriction.i did plasmid mini prep of e coli protocol. In gel my plasmid is not moving from well. I thought this protocol is not much good for Agrobacterium. so can any one provide me agrobacterium mini prep protocol which is not kit based.
Thanks in advance.
SVRREDDY,
Graduate student.

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You normally can't get enough DNA out of agrobacterium using an E. coli miniprep to do a restriction digest. There are a couple things you can do instead.

1. Do an e.coli miniprep, then take whatever DNA you did get, transform E. coli with it, then proceed to grow up the E. coli and test it with your restriction digest. This is more time consuming, but generally consistent.

2. Do PCR direct from your colony, including a non-transformed colony as a negative control. Include a 5 min boiling step before the actual PCR to break open the cells. This is much easier and what i've been doing lately, although you really need to include the negative control to make certain that the PCR product is real and not contamination.

I do have a protocol for doing a miniprep direct from agro, but it has never been consistent for me. Try one of the above two methods first and if you don't get what you want, then let me know and I'll send you the protocol.

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