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Trouble getting qiagen DNA

Trouble getting qiagen DNA - Protocols and Methods Forum

Trouble getting qiagen DNA - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 01-04-2005, 08:45 PM
PS
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Default Trouble getting qiagen DNA



I am working on a 11kb plasmid and trying to get a qiagen prep of it. I
have tried twice once using high-copy plasmid protocol with qiatip-110
and once using low-copy plasmid protocol with qiatip-100. Both times I
did not get ANY DNA from the preps. I know that my techique is right
because I ran other plasmid preps parallel to this one and got tons of
DNA for it. Hence I think the problems that I am encountering are
specific to this 11kb plasmid.

Let me tell you more about what I think.

1. This plasmid grew well in LB AMP (100ug). This was the culture I
used to prep qiagen DNA and got nothing. I streaked a plate from this
culture and suprisingly nothing grew. I always thought that liquid
culture is more stringent than agar plate and it is very surprising to
see that the culture that grew in liquid AMP did not grow on the plate.
May be that is the reason I did not get DNA but how come the cells grew
in liquid culture
2. I also don't think that this plasmid has gone really low copy
because I have a miniprep from 1.5ml of the original culture (the
culture that I talked about above was scaled up from this original
culture). I digested 3ul of this miniprep and I could see good amounts
of DNA on the gel. So my guess is that somewhere in the middle the
plasmid is lost.

I have plans of retransforming the miniprep I have, testing that to see
if it has the plasmid by digesting and then use that retransformed
cells to scale up my culture for qiagen.

It would be great if I can get more suggestions to get this 11kb
plasmid DNA

Thanks

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  #2  
Old 01-05-2005, 02:03 PM
J Winkler
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Default Trouble getting qiagen DNA

PS wrote:
It is generally considered to be the opposite. Selection on plates is
more effective than selection in liquid cultures.

In any case, it is a good principle to always start from a single colony
from a freshly streaked plate, do a preculture & then inoculate the
midiprep from that starter culture.
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  #3  
Old 01-06-2005, 12:18 PM
Bas Jansen
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Default Trouble getting qiagen DNA

In article <1104871515.292554.95840@c13g2000cwb.googlegroups. com>, PS
<[Only registered users see links. ]> wrote:


We have had some problems in our lab regarding constructs containing
genomic DNA. We never cared to figure out what was the problem, as we
went back to the good 'ol cesium chloride gradient protocol. With great
success, I might add.

Bas
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  #4  
Old 01-06-2005, 04:29 PM
Dr. Hiranya S. Roychowdhury
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Default Trouble getting qiagen DNA

The basic principle on which the regular Qiagen columns are based does not
allow good yield of very small to large size DNA's. You will have a better
luck trying their free suspension of the silica. The columns will shred the
11kb plasmid. Also, I find it immensely better to prepare plasmids through the
conventional alk lyses, and the yield is between 2 to 5 fold higher than any
kit.

Quoting Bas Jansen <[Only registered users see links. ]>:



--
Hiranya S. Roychowdhury, Ph.D.
Coll. Asst. Professor,
Molecular Biology,
Dept. of Chemistry & Biochemistry
Rm# 336, Chemistry Bldg.; MSC 3MLS
New Mexico State University
Las Cruces, NM 88003
---
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  #5  
Old 01-06-2005, 07:52 PM
Bas Jansen
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Default Trouble getting qiagen DNA

In article <[Only registered users see links. ].edu>, Dr. Hiranya S.
Roychowdhury <[Only registered users see links. ]> wrote:


In all fairness though, I have to say that we generally haven't had
problems with constructs at around 10-12 kb and Qiagen columns. With
'no problem' I mean that we got the expected yields indicated by
Qiagen, not the yield that is to be expected with cesium chloride
gradients (which is in my experience far higher, and cleaner). Only a
few, which upon closer inspection also contained a lot of genomic
repeats, gave us trouble. But I agree with you that conventional
protocols sometimes are just plain better, yield- and quality-wise.

Bas

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