colony PCR

[Michael Sullivan]

To do PCR on agro, I usually grow up a liquid culture. Harvest 100 ul  
of culture by centrifugation, resuspend in 20 ul water, then boil for  
10 min. Spin out debris for 5 min. I use 5 ul of this in a 25 ul PCR  
reaction (which is probably a lot more than needed).

This procedure works well for me for detecting genes in the Ti-plasmid.

Usually for checking my binary construct I do a miniprep and check  
the digestion pattern. I prefer this since it gives you more  
information than just amplifying a portion of a gene (i.e. digests  
might tell you you've had a rearrangement, etc.).

I've had good luck doing Qiagen minipreps with agrobacterium. Quality  
of the DNA is usually good enough to work in a restriction digestion.  
I've had more variable results with standard minipreps: digestion  
with these sometimes works, sometimes doesn't. If you make DNA from  
agro and your digests don't work, the DNA  is usually good enough to  
retransform into E. coli. Plasimd can then be prepped from E. coli  
for analysis. One thing to keep in mind with DNA from agrobacterium  
is that methylation is different and may effect some restriction  
sites with overlapping Dam methylation (e.g. ClaI).

Doing restriction digests may take a bit longer than doing a quick  
PCR reaction, but compared to how much time will be spent on plant  
transformation, I think it's worth spending a few minutes to make  
sure the the binary plasmid in your agro is correct.

Mike

On Mar 14, 2008, at 12:57 AM, Sunil Dalvi wrote:

> I am in need of colony PCR method . I got the positive colonies for  
> Agro but
> not getting the band for insert. I got the band for positive  
> control in the
> same reaction.
> where would be the problem
>
> -- 
> S.G.Dalvi,
> Vasantdada Sugar Institute,Pune
> Manjari bk, India
> http://www.vsisugar.com/
> _______________________________________________
> Methods mailing list
> Methods from net.bio.net
> http://www.bio.net/biomail/listinfo/methods

---
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
ARS-USDA
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)