Mouse Tail Genotyping: Viscous DNA - (Oct/24/2008 )
I have been troubleshooting the mouse genotyping protocol in our lab for a few months now. We finally got it working well and then, on this last round of genotyping we ran into an issue. The DNA precipitated well, but, upon resuspension, it was very viscous and stringy. This has not happened in the past, and we had actually noticed that even the aqueous layer was a bit viscous before the addition of ethanol.
The problem is that my housekeeping gene did not amplify in PCR. I saw results with my other two genes of interest, but I got nothing from the housekeeping gene. Could this be because of the viscous DNA? I also notice that many protocols say not to use viscous DNA, does anyone know why? And last, does anyone have any suggestions as to why this may have happened and what I can do with the DNA now to get it to work.
I am using the standard chloroform:phenol: isoamyl alcohol method with an ice cold ethanol precipitation..
Thank you so much!!!!
perhaps your concentration is too high? possibly it would help to run a couple 1:10 dilutions and see if you can get a better result.
what is the buffer you used for resuspension?
try to dilute your prep, and pass it through a QIAgen column to get a homogeneous solution
How big are your pieces of tail? I use teeny tiny bits (about 1-2 mm), and find that they give more consistent results than big pieces. As others said, try diluting your DNA 1:100 or 1:1000 and PCR again.