I am doing agarose gel electrophoresis of bacteriophage (lambda DNA), and getting inconsistent results for single stranded bands. In my experiment denaturation of lambda DNA is done using 50mM NaOH (in solution) and incubating the solution at 37C for 60 min. 1% agarose is used along with 0.5% TAE buffer for running the gel. Agarose used (product no. A2929) is supposed to be good for long DNA. I am unable to figure out the problem with my experiment. So if anybody have any experience of single stranded lambda DNA electrophoresis, any explanation of procedure/outcome would be greatly appreciated.
If you run denatured DNA in the non-denaturing gel such as TAE, I would assume DNA will be partially renatured, and form a 'mesh'-like structure, and perhaps will be stuck near the well.
To avoid this, you can simply use alikaline agarose gel.
However, I am not sure if you can separate 40-kb single-stranded linear DNA, even if you use alkaline gel. (You can try.)
In addition, such a long ssDNA will be highly fragile, and will be easily torn while pipetting. You will need an extra care in handling.
Hi Shin-Ichiro,
Thank you for your input.
The denatured DNA bands I am getting are "very smeared " and are behind the dsDNA bands.
I am also not sure if separation of such a long ssDNA using a gel is possible or not. Even though, I will try denaturing gel soon.
I am aware that such a long ssDNAs are very fragile and need an extra care in handling.
Hi Shin-Ichiro Hiraga,
Thank you very much for your advice. The separation of single stranded lambda DNA is possible using alkaline gel electrophoresis.
- Badges
- Science method