After extracting total RNA from tissue using trizol reagent and phase lock gel,h - I am going to extract bothe total RNA and genome DNA from one sample (Aug/02/2007 )
Hi,all
I am going to extract both total RNA and genome DNA from one sample ,after extracting total RNA from the tissue using trizol and phase lock gel ,I don't know how to extract the genome DNA after using phase lock gel ,can anyone give me some help?
hi, can anyone help me ?
well... after using trizol, you'll gona have rnas only in aqueous solution.
then you need to pipett the lower phase.
Precipitate it with buthanol (as the phenol is greatly soluble in BuOH).
Resuspend in appropriate volume of water/TE/10mM Tris pH 8
add the phenol chlo isoamyl alcohol mixture (pH 8 this time)
spin and proceed as usual for DNA extraction.
Careful !
no vortex, only hand shaking in order to minimize DNA shearing.
then you need to pipett the lower phase.
Precipitate it with buthanol (as the phenol is greatly soluble in BuOH).
Resuspend in appropriate volume of water/TE/10mM Tris pH 8
add the phenol chlo isoamyl alcohol mixture (pH 8 this time)
spin and proceed as usual for DNA extraction.
Careful !
no vortex, only hand shaking in order to minimize DNA shearing.
thank you for your help!
so I just pipett the lower phase under the phase lock gel, then use this liquid to extract genome DNA?
and anothe question is vortex will shear the genome DNA , so , will the homogenization shear the genome DNA too?
thank you!
well i used to homogenize with a 1000µl tip.
So i guess there my be a risk at pipetting, but at this time i don't truly see the trick.
Alternative : you may separate your tissue into 2parts and extract with DNAzol one half and trizol the other half....