Qiagen: This is ridiculous

Pekka Rappu TUY prappu at finabo.abo.fi
Mon Sep 2 09:04:27 EST 1996


I have tried to figure out the reasons for occasional poor or no yields of
Qiagen gravity-flow based midipreps. I have noticed that the mixing after
addition of buffer P3 is very important step. If the mixing is insufficient,
SDS doesn't precipitate well, which affects the binding of DNA to the column
matrix. At least with 50 ML Oakridge tubes 6-8 quick inversions have 
usually been enough. 

The second thing is the growth medium. I have had best results (100 ug or
even more, purity: GS-rich DNA sequenced about 600 bp or more with ABI 377
automated sequencer) by using medium containing tryptone 30 g , yeast extract
20 g and MOPS 10 ug in 1 liter, pH about 7. For high copy plasmids I have
used 50 ml of culture per column and 200 ml for low copy plasmids (or low
copy strain). For 200 ml I have used 10 ml of buffers P1, P2 (made freshly)
and P3 instead of 4 ml. Because of larger volume, I have extended the 
incubation time on ice to 20 min. The strains I have used are DH5 alfa 
and KE94 (low copy strain). 

Thirdly, I have noticed that the culture should be grown for more than 15 
hours. This can be a case spesific thing, but when I have grown the 
culture for about 12 hours I have had poor yields.

Maybe this helps somebody, maybe not. It's funny that the people at Qiagen
have spent months or maybe years in optimizing the kit and then you have to
do it all over again. If you want to try the slight modifications above 
please let me know how they work in your lab.

-Pekka





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