Help Needed! DNA sample dispersed b4 electrophoresis - (Oct/09/2005 )
I have extracted B. subtilis DNA using by alkaline lysis and precipitate using isopropanol and ethanol.
I even have completed dried the ethanol with heat-drying speedvac.
however, when I mix with gel loading buffer and load my sample into agarose gel well, the sample sank at the first place. But as soon as I remove my pipette from the gel, the trace of gel loading buffer with my sample started to dispersed into the buffer. It seemed like a thread unwind from top till the bottom of the well.
I thought my sample contained ethanol or some other contaminants that might cause all these. So, I freeze-dried my DNA sample again and I still face the same problem.
Was this due because my DNA sample was too viscous? The concentration of my DNA is very high.
Please help me!
That would be my first guess... Dilute it 1:10 or so, and try again?
What was the original culture volume, and what volume have you resuspended the resulting DNA in?
Original volume of culture was 100mL with OD of around 2.5. The reference method that I have used suggested OD of around 1.0 only.
After speed vac, I resuspended into 4 tubes. Then I freeze-dried the four tubes again. I resuspended the four tubes, each around only 300 microL with tube 2 around 500 microL.