Strange result with DNA assay - (Aug/23/2006 )
I tryed a different assay in order to see DNA fragmentation.
I use DNAsol, from invitrogen, to extract DNA from muscle tissue from fish. I used cold ethanol.I used NaOH 8mM to resuspend it.
I put the pure DNA on an agarose gel 0,8% and let it run for 40´on 100v.
I got really strange result:
The DNA from the fish control (from clean water) run perfect and i got a really nice band.
The DNA from the fish with I was testing, no! It didn´t let the pool! I tested 10 samples and wich all of then, except in one, I got the same result: dna on the pool.
In just one sample, I got something like a small smir.
I couldn´t understand these result! I was waiting to get big smirs from all the samples, except the control one.
I try to find something on internet, I asked lots of people, but nobody could help me.
I am thinking about some possibilities:
-The samples have to much protein: but way the control run perfect? (I use exact the same metods for all the samples). Where did that protein comes from? Because I used the same part for all fish!
-If the DNA is fragmentated, way it didn´t run?
I am thinking about trying two new metods:
1- Run the samples on a 1,5% agarose gel on 40V for 3 or 4 hours.
2-Heat (94ºC) the samples and then let they run on a 0,8%gel for 40´100v.
thanks a lot!
If the DNA is already sheared before or during the extraction method you will end up with a pellet of small DNA fragments that you resuspend in NaOH, you will end up with very small degraded DNA
have you tried to resuspend the DNA pellet in water, measured the OD260 (nanodrop or spectofotometer) then run it on a agarose gel what do you see then?
If your DNA is still intact, then you can fragment it with NaOH,
but I prefer to use DNase / sonication or heat