pulsed field gel electrophoresis of genomic DNA - (Feb/12/2008 )
I am currently growing a poxvirus in eggs - when I extract DNA from the harvested virus there is always a little bit of chicken contamination. I want to do a PFGE to try to separate the virus (300kb) and chicken (1Gb) DNA. I am unfamiliar with PFGE protocols and was wondering if it is ok to just load a straight DNA sample into the well or do I need to use an agarose plug? Any help would be much appreciated - also if anyone knows of a protocol/basic method for this type of separation. Thanks.
To get a clean separation you will need to make cell plugs. Anything less, and you will shear your DNA into fragments.
The plugs should be made with low melt agarose, low EEO. The plug should be made to a final density of 0.6% agarose in PBS buffer
Have you cell sample.
- Concentrate your cell sample to X cell/ml in PBS buffer... different cell types require different amounts of cells to give a nice picture...I don't work on chicken cell or poxvirus. You will have to figure out that proper concentration yourself
- To the cell sample, add equal volume of Low melt agarose (in PBS).
- Add mixture into plug caster.
- Leave on ice to set.
- Meanwhile add pronase powder to NDS buffer to a final concentration of 1 to 2 mg/ml pronase.
- Eject plug into a small container (such as a 15ml fulcon tube). Cover the plug with the Pronase/NDS solution
- Incubate in a water bath overnight at 55 Celsius
NDS buffer
0.5M EDTA pH 8
0.01M Tris pH 8
1% N Lauroylsarcosine
Once you have your plug add it into the plug gel. And let it run.
1.5% Agarose in 0.5xTAE.
Buffer = 0.5% TAE.
I think a pulse time of 45sec at a constant voltage of 150V at 10 Celsius, overnight run should do.