DNA purification

[Silvia_84]

Hi all,

I have a few questions about the purification of DNA. I followed a protocol specified in a kit for the DNA extraction and purification. The last step of the procedure says to rehydrate the DNA in a rehydrating solution (already present in the kit). I have done it but I have difficulty to dissolve the DNA and make it go in solution. I am able to see the DNA pellet. My supervisor suggested me to flick the tubes just with my finger to try to dissolve the DNA..but the pellet is still not dissolved.Can you suggest me something to get the DNA in solution?
My second question is: how can I determine if the concentration of DNA (after purification)is good and significant (in terms of amount)for next experiments? Are there some range of values of concentration to consider as good for the DNA?
Is it better to diluite the DNA before using them? Which dilution? 1:10?

Thank you very much in advance.

Silvia

[CharonY]

Hard to tell, without knowing the specifics of the protocol. However, it sounds like overdrying of the pellet. Is the pellet opaque or whitish? Things to try are either let it sit in buffer for longer (in the fridge) or with mild agitation (shaker). If you are not worried about integrity (e.g. for most PCR) you can even do mild sonication.

 

With dilution you would need to know your concentration and what your application calls for. There are no universal values.

[Arete]

Personally (I'm never usually worried about integrity, as I'm either going to PCR it or shear it up for a short read library) I hit it with the benchtop vortex for a minute or so, or I leave it a room temp on the bench for an hour or so. Another possibility is that the pellet you're observing is actually contaminants that the purification step failed to remove, rather than DNA. Depending on the tissue type, it's sometimes impossible to get all the contaminants out of an extraction with a standard protocol (I'm looking at you, jellyfish samples. Grrr.).

[Xalatan]

Maybe there is still residual ethanol from the precipitation step that's preventing dissolution.