Why we must use freshly prepared NaOH/SDS for miniprep - (Feb/06/2006 )

Why we must use freshly prepared NaOH/SDS?what is the purpose of preparing this solution fresh?

Hi-

You're talking about the NaOH/SDS solution for minipreps? I actually don't make it fresh myself. I think the logic for making fresh though stems from the fact that it precipitates pretty easily. I forgot and put it on ice once and it precipitated right away. I've heard that you can just warm it up and it should just go back into solution but I've never tried it myself.

Mountainman

The solution will begin to acidify due to carbon dioxide in the air reacting with the NaOH, in theory this will diminish the denaturing power of your solution. In practice, I find solutions can lyse cells effectively for upwards of a month. Just be sure to keep them well capped. As an aside, has anyone ever tried doing a restriction digest on the crude plasmid pellet, i.e. before phenol/chloroform extraction? Cheers.

I never P/C extract mini-preps; if I need really 'good' DNA I don't use a mini-prep, I only perform them to check clones

after the PIII step (the NA/oAc precipitation), do the spin, remove the supernatant and add it to an equal volume of ISOH. spin for 30' at 4C. air-dry the pellet thoroughly. resuspend as usual in 50ul TE and set up a digestion

I've been doing it this way for years and it works pretty well; it is also 'clean' enough to add 1 ul to a transformation if you need to make more

Amikins, you don't do an ethanol wash after the isopropanol ppt?

I though you were supposed to do that.

The NaOH/SDS solution that ships with kits is likely months old, and it works fine. It is not exposed to the atmosphere, though -- so I guess statler's point is valid. Though we buy miniprep kits at 100 preps per box, which lasts us quite a while after it's opened, and we've never had a proplem.

I usually don't do EtOH washes of ppt DNA either... I think the point of an EtOH after an isopropanol ppt is because isopropanol pellets are frequently somewhat hader to dissolve that ethanol pellets, and ethanol is more volitile than isopropanol, so you can evaporate it off quicker.

I know that salt reduction is a frequently cited reason for EtOH washes, but I've never had a salt problem, so I don't usually bother with them...

well, basically, if I want the pellet to dry faster I would use an EtOH wash. frequently I am not in that much of a hurry (letting the pellets dry at the end of the day or something anyhow) and it doesn't take that long to resuspend an ISOH pellet; I did this the other day with a big batch of minipreps. I let it dry overnight. came in the next morning and added my 50ul TE. by the time I was done writing up my cocktail recipe for the digestion to check the clones, they were plenty dissolved.

Now, I am not saying this is the cleanest DNA, it's just easy and it saves you some steps. it digests just fine (no smears or inhibition of the enzyme or anything like that)

I have never done subsequent cloning or sequencing (of course!) without a little further purification, though you can digest, PCR, transform, whatever you gotta do to confirm the clone

I also routinely use miniprep to chech clones. When I digest I add some RNase
because small RNAs may mask bands on agarose gels.

Good luck.

it won't "acidify" but you will get some precipitation of the carbonate salt.