problem with plasmid extraction - (May/08/2009 )
i am using Lactococcus lactis bacterial strain,which contains the 8kb plasmid.i used nuclegen plamid exraction kit.but problem is i am getting very less yield and when i am trying to run in gel,if i take small volume i.e 1-3 microliter nothing can be seen in gel....so i tried taking 10 micro liter..but i see faint band...is there any thing i can do about getting more plasmid yield...
Have you tried qiagen plasmid extraction kits?
There are three types i think depending on the wanted/expected yield of the plasmid and also on the size.
Is it possible that the extraction kit you are using only offers the type of yield you are getting?
Maybe try the qiagen midi or maxi extraction kits.
Also have you tried to quantify using pico green or similar methods to determine exact amount of plasmid extracted?
Inoculate multiple tubes and pool the yield.
I am having similar problem. Having very low yield of maybe 15-30 ng/ul after using up to 25 ml of culture and heating up the elution buffer to 70C for elution. I am using miniprep to extract the plasmid after incubating the cells with lysozyme for 30 min at 37C.