5 replies to this topic

[vincio]

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!

Edited by vincio, 17 March 2012 - 03:32 AM.

[hobglobin]

perhaps not enough mitochondria available in blood?

[vincio]

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...

[hobglobin]

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.

[vincio]

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

[hobglobin]

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.