Particle bombardment

Michael Sullivan mlsulliv at wisc.edu
Tue Nov 15 14:36:08 EST 2005


Is the concentration you are trying to achieve a typo? If not, there  
is some major problem because you should be able to get well over  
that concentration with a commercial miniprep kit, even for a lower  
copy number plasmid.

I use Qiagen columns for minipreps. My typical yield from 2 ml of  
overnight culture in LB is 50 ul at 200-500 ng/ul for high copy  
plasmids. Even with a low copy plasmid, I think you could expect to  
get DNA at a much higher concentration than 1 ng/ul.

I can think of one possible problem with your preps, if you are using  
Qiagen minipreps. Are you sure ethanol was added to the wash buffer  
PE. At one point, an undergraduate student in my lab was getting VERY  
poor yields on minipreps. It turned out she had not added the ethanol  
to the wash buffer so (I presume) most of the DNA was being washed  
off the column.

It would be useful to know more information. What is the plasmid  
vector, selection, how big, how do you grow the culture.

Mike



On Nov 14, 2005, at 4:40 AM, Sharp Tool wrote:

> Hi,
>
> Normally a 0.5 - 1.0 ng/ul of purified plasmid DNA is used for  
> particle bombardment.
>
> QUESTION: Does anyone use a commercial miniprep kit to purify  
> plasmid DNA from an overnight culture to achieve this  
> concentration? I found that the yield is very low, and no way of  
> getting this concentration unless I precipitate the DNA and  
> resuspend it in a smaller volume.
>
> Sharp Tool
>
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>

---
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)

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