Help! The yield of my large-scale plasmid DNA is low!

Wolfgang Schechinger Wolfgang.Schechinger at med.uni-tuebingen.de
Tue Jun 1 07:48:46 EST 1999


Wilson, 

Well, 2 mg is not contradictory to "up to 10 mg"...
Overloading will reduce your <yield, because some other stuff also 
wants to bind to the column (you do a fractionating elution), thus 
you'll get less DNA. I case of doubt, measure the OD660 of you 
buggers, feed them the right media and just treat them as told in the 
manual. I know, following a manual can be tedious, but in this case, 
you can't escape. That's my own experience.

If you don't find another cause of your prep failing (low 
expression, column overload, I'd blame the pH of Buffer P3). Did you 
preserve samples from each step for quality control? Then you should 
see where the devil's inside.
A second elution step with the same volume of elution buffer might 
incerease your yield.

I am re-using my maxi columns from Macherey-Nagel (same as 
Clontech's and also ion exchanger based like Quiagen's), too with no 
apparent loss of yield (5 times yet). MN claims that one may directly 
re-use the coumns without further treatment after eluting the plasmid 
(just re-equilibrate and start over). I am washing my columns with 50 
ml of 2 M NaCl and then with 100 ml of ddH2O, afterwards I let 
them dry before storing them in a plasic bag. Then I start again with 
the equilibration step. 

Did quiagen tell you the composition of their buffers? That I 
couldn't elucidate this was one reason (beneath the price) to switch 
to Macherey Nagel's columns (they might not have a Giga column on 
the shelve, but you could inquire anyway).

Good luck! 
Wolfgang
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Wolfgang Schechinger   
Pathobiochemistry Dept.      
University of Tuebingen, Germany
email: wgschech at med.uni-tuebingen.de * wwWait: http://www.medizin.uni-tuebingen.de/~wgschech/start.htm
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