mtDNA extracted from hair is coming up degraded...why? - (May/13/2007 )
i've been extracting mtDNA from hair shafts, using a protocol which has worked before, however, when i've tested the pure dna with gel electrophoresis, it's coming up smeared...ie degraded. Does anyone know why this could be...the hair i'm using is dyed, coul dthis have an affect on the stability.
Which method do you use? I guess it might be Dnase contamination.
I digest the hair in DTT, Tris, Proteinese K, CaCl, Water, NaCl, SDS overnight until all traces of hair is dissolved. then vortex and do organic extraction twice with Phenol:Chloroform:Isoamyl-alcohol, then ethanol precipitation, and ethanol wash, then let air dry and rehydrate with 50 microliter of TE buffer.
Now the hair I was using, was "cut" and from an individual who dye's her hair regularly...do you think this may play a part with the degradation?
Thanks
"cut" hair? Erm... I am not too sure about hair though. But when you said "cut", is the root hair included? DNA is extensively found there instead right?
For hair dye, yea, it might possible to have some sort of DNase contamination. But again, with DTT, it shouldnt have any activity. 
For hair dye, yea, it might possible to have some sort of DNase contamination. But again, with DTT, it shouldnt have any activity.
you're right, nuclear DNA is found within the root portion, however, due to an extensive apotosis like practice the cells in the hair shaft contain quite alot (or should) of mtDNA. I too thought that with a good allotment of DTT, it would counteract any contamination from chemical treatment, but i'm wondering if the dye used on the orginal hair, containted bleach and or ammonia, which might have penetrated the shaft, thereby degrading the DNA. I am trying the experiment again, this time with dyed hair, but instead of "cut", hair that was combed, so it may included longer pieces, as well as root ends. Thanks for the help
Not really much help anyway. Hmmm, perhaps you can try DNAse inhibitor?
Another question, did you get any string of DNA when doing ethanol precipitation? 
Another question, did you get any string of DNA when doing ethanol precipitation?
DNAse inhibitor...good idea. When I did ethanol precip, i actually got a nice pellet, so i guess we'll see how this new attempt goes
Good luck with it. If you are getting nice pellet? The Dnase shouldnt act that quick. Let us know about your latest attempt. 