Maxiprep - (Oct/29/2007 )
I am having problems doing a maxiprep of my plasmid. I have been using the QIAGEN kit with the filter columns and when I follow the protocol it all seems fine, and all of the samples I take out run as expected on a gel, but there is no pellet at the preceiptation stage.
I tried a "normal" maxiprep (using solutions I, II, III) on the same culture and obtained lots of DNA, although it is too dirty for the purposes I need it for.
What could I be doing wrong in the maxiprep? Is there another way to do the precipitation which might have better results?
Hi,
in our lab we distribute the elution (after adding isoprop and mixing well) into 1.5ml Eppis and spin in those ones. It`s defininitely easier to see the pellet. We use the midiprep kit but it should work for you as well. If you still don`t see any pellet maybe it`s something with the bacteria.
Cheers
On commenting on the 'normal' maxiprep, it is important not to shake the cell solution after adding solution II. A single gentle bit of swirling is okay, but not any more. The cell pellet should be suspended in a large enough volume of solution I (cell concentration) in a wide enough container (tall containers make mixing difficult), so that on adding solution II, the mixture almost mixes itself. Leave the mixture standing in solution II, for a few minutes (the duration depends on the mass of the cell pellet. However leave it too long and you get too much denatured DNA). After that add solution III. Again don't be too rough, a little gentle swirling. And once the mixture is neutralise, then you can act more 'normal'.
Works perfectly fine for BAC from 30kb to 190kb.
Well, one loses a lot of DNA with the columns. Unless its a recent problem, try to do a maxi with 200ml O/N culutre, it can help your chances. Also is the plasmid high copy or low copy, this too has to be taken into consideration. Try a different kit, if you feel it could the kit.
Have you resuspend your pellet (i know you dont see one) anyway and run it on gel or check OD? Maxiprep pellets are pretty much transparent, and sometimes difficult to see, also, do you not see a pellet after isopropanol, or after the Ethanol wash. how are you spinning down your pellets and removing the buffers, is easy to lose the DNA if you tip the tubes over. when using corex tubes i found that marking where my DNA should be help loads, remove IPA, wash, remove EtOH carefully, dry a bit, resuspend in ~200ul TE or H20, run on a gel. Many many times I havent seen a pellet, but lovely clean DNA on gel.
Whiteness in pellets is due to salts, columns clean your DNA very well (also reducing yields thats true) so your DNA becomes clear.
Have you resuspend your pellet (i know you dont see one) anyway and run it on gel or check OD? Maxiprep pellets are pretty much transparent, and sometimes difficult to see, also, do you not see a pellet after isopropanol, or after the Ethanol wash. how are you spinning down your pellets and removing the buffers, is easy to lose the DNA if you tip the tubes over. when using corex tubes i found that marking where my DNA should be help loads, remove IPA, wash, remove EtOH carefully, dry a bit, resuspend in ~200ul TE or H20, run on a gel. Many many times I havent seen a pellet, but lovely clean DNA on gel.
Whiteness in pellets is due to salts, columns clean your DNA very well (also reducing yields thats true) so your DNA becomes clear.
I will try again today and use the smaller tubes and mark on the tubes as suggested. What other kits are there for DNA prep of this kind? I need it to be very clean as it's for embryonic stem cell injection.
I agree with almost a doctor.
I don't have a lot of experience with different kits, but it is often difficult to see the pellet with these kits.
When I do a "dirty" extraction (only solution I II and III and no column, just to digest with efficient RE and check on a gel) I see clearly a pellet, because of the salts.
So, no pellet, good news, your DNA is clean.
You could try the Macherey Nagel kits, they work perfectly well and are not so expensive as Qiagen. I would also try to use new Isopropanol and Ethanol solutions, we once had a problem when the ethanol was too old. And mix your eluted DNA very well with the isoprop, otherwise you will never get a pellet, it will just form phases.
I also have had terrible luck with the Qiagen maxiprep kits, for reasons I do not understand. The minipreps work great, and one would think that the maxi is simply a scale up. The kit which works for me is the Biorad Quantum maxiprep (NOT the Aurum maxiprep kits, which behave very much like the Qiagen ones).
We manage to get good yield from the invitrogen kit and also Macherey nagel.