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mitochondrial DNA extraction from avian blood -urgent - (Mar/17/2012 )

Hello,
i am working whit philogenetics whit mitochondrial DNA dloop sequencing on poultry. I am using a rapid DNA extraction from whoole blood whit two methods:
a)
1. wash blood whit TE buffer 2 time
2. digesting whit proteinase K and PCR buffer

or

1) 1ul of whoole blood in 300 5% chelex resin
2) incubated 1 hour at 60 C and 15 min at 95

Both methods work excellently for microsatellites analisis but when i used this DNA for mitchondrial DNA I did obtain any amplification. I use a positive control extracted whit phenol-croloformium mehtod and I obtained strog amplification both from microsatellites locus as mitochondrial fragments.

So my question is...could rapid methods be not enogh to lyse mitochondrial membrane?

Tank you!

-vincio-

perhaps not enough mitochondria available in blood?

-hobglobin-

mmm I don't know the mitocondria nember per cell in avian but if it is similar to mammalian must be more than 800-1000...

-vincio-

Well I'm not an expert about vertebrate blood (I work with blood-free animals), but you can only employ leukocytes to get mtDNA, right? Perhaps the cell number is too low to get enough, that was my idea.

-hobglobin-

thanks, but is exactly the opposite in chicken: they have eritrocite whit nucleus and mitocondria, so the number of this organelle may be high. The strage fact is that the extraction whit phenol-cloroform work good for mt dna analisis...the fast methods only for microsatellites

-vincio-

Okay, didn't know that...you might try out to add SDS or Triton-X 100 to the buffer of the fast methods, if it works then, membranes seem to be the reason.

-hobglobin-