problems with qiagen preps.

Basavaraju Shankarappa bsh at MED.PITT.EDU
Sat Jul 9 12:11:44 EST 1994


VOSS at AIMP1.UNA.AC.AT wrote:
>: Hi out there,
>: Did anybody else out there run into similar problems?
>: Looking foreward to your experience
>: Tilman
>
>I'm sorry if I missed an earlier posting describing the problems that you 
>are having with the Qiagen preps, but I've used Qiagen preps since they've 
>been out and the only problem that I've ever had was when the buffers were
>old and the pH had changed. They *do* warn of this potential problem, but 
>usually the kit is gone before there is a chance for this to happen. 
>Alternatively, if you make your own buffers instead of buying the kit, pH
>of the buffers may be the issue.
>The other difference that I've noticed in the latest versions of the Qiagen
>method is that they switched from ethanol to isopropanol precipitation so 
>that very often from large scale preps you may not notice a precipitate or
even
>a pellet at the end. The DNA is usually there upon resuspension and
subsequent
>OD260 and gel analysis, but when you're used to *seeing* a pellet from large
>scale alkaline lysis preps, it can be a shock. 
>
>I hope that I've hit on what you may have been asking about. If not, please 
>repost or alternatively send me e-mail. Perhaps I can help.
>
>Ellen
>
>E-mail: ellenmq at bronze.ucs.indiana.edu
>
I have experienced quite a lot of problems specially with respect to 
variability in the DNA yields.  The two attempts I made using Qiawell
resins worked very very beatifully.  So we thought this looks like a 
very useful and easy method to prepare DNA and we went ahead bought the
kit, which is not necessarily cheap.   But the next three attempts using
the same reagents and same batch of plates was a disaster.  Some of the
filtration steps were also very cumbersome and the yield was very low.
So now, I came back to good old PEG ppt outlined in the DyeTerminator manual.
The yield and sequence read is not very great but atleast, it gives very
consitent sequence reads.  
Raj Shankarappa
bsh at med.pitt.edu
 



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