genomic DNA extraction - (Sep/06/2005 )

we tried to extract genomic DNA from tissues (fresh and frozen) using phenol and salting out protocols.
running the DNA in the gel showed us - smear. we changed the water, reagents, we also washed the tissue with sPBS or ethanol before lysis.
another thing is that we haven't seen the DNA as a thread during adding 2V ethanol and 0.1V salt.
the phenol protocol worked once few years ago - for blood and now we are using it for brain, muscle and ovary tissues
can anyone have other ideas?
thanx

smear is indicative of DNA shearing and I would expect that from frozen samples.

unless you have several micrograms of DNA you won't see the threads of DNA upon precipitation.

on a gel are you not seeing any DNA at all?

Nick

What are you doing with this genomic DNA? For most applications, such as PCR, this DNA will probably be fine. Very high molecular weight DNA will typically be stuck near the well. If it is fractured by vortexing or pipetting through small orifice tips, the DNA will show as a smear. This will likely not affect most use.

1. i need the DNA for southern so that i need good extraction.
2. in the gel i saw a smnear (not overloading)
3. i used up to 1 g of tissue and still haven't seen the threads .
thanx a lot

We use the Gentra kit after encountering the same problems. More than anything - you cannot vortex the samples (even if the proticol tells you too). Inverting multiple times seems to be enough to mix our reagents successfully. If you don't want to buy a kit, try modifying =your protocols to make them gentler and don't freeze thaw your DNA more than twice.

does your sample subject to any heat treatment? this is to get rid of DNase