troubleshooting low yield midiprep - (Jul/17/2006 )
I´m trouble because after grown a positive colony of ligation (5Kb plus 2.2 Kb) I took an alicuot of LB but I didn´t obtain a standard concentration of DNA with purification plasmidial DNA Kit (promega). I obtained 0.2 ug/ul instead of 1 or 2 ug/ul. I don´t know what do I do to improve it?
Aloaiza 
What do u mean by taking an aliquot of LB? How much of the LB (overnight culture) did u use to extract DNA?
I get 1-2ug/ul only when I do maxi preps.
if u want to get high amount of DNA like 1ug/1ul u must do large prep. i usually do large prep by CsCl gradient and i get 2-6ug/ul DNA
What I do after a midiprep is to precipitate the DNA and resuspend it in a smaller volume of
buffer which gives me concentrations of up to 1,9 µg/µl
buffer which gives me concentrations of up to 1,9 µg/µl
do u get pure DNA (A280/A260= 1.8 to 2.2) by that way??/
What I do after a midiprep is to precipitate the DNA and resuspend it in a smaller volume of
buffer which gives me concentrations of up to 1,9 µg/µl
do u get pure DNA (A280/A260= 1.8 to 2.2) by that way??/
After precipitation, I usually get pure DNA - 1.8 - 2.0
oh thats great , how long does it take to perform midiprep???
It takes me 2hrs to do a maxi (invitrogen). I havnt done midi preps.
by this way how much DNA do u get?? that is the amount of DNA . like from large prep by CsCL gradient i get 400ug~ DNA from 50 ml culture
I get around 1-1.5mg of DNA from a maxi prep - 250-500ml culture. Depends on the plasmid. Some times even upto 2mg.