QIAGEN Spin Columns and Minipreps - Alternatives?! - (Aug/15/2012 )
i still do my minipreps the old fashioned way, just add a chloroform extraction and PEG pptn step to get sequence quality DNA. It really doesn't take much longer than the kits, as you can do other things during the precipitation steps.
Also note that buffer p2 on the openwetware page should be made fresh each time as the NaOH gets neutralized by CO2 absorption from the air.
I also used to work in a lab that made their own (Promega) wizard miniprep columns using cheap filters and diatomaceous earth. I can't remember what sort of preparation was required though.
ascacioc on Wed Aug 15 16:00:03 2012 said:
I personally believe that the only thing worth buying from the kits are the silica columns. Anything else is just simple buffer you can make yourself. Buying a TE buffer from a company is a total waste of money.
In the old times (like 10-15 years ago) they did not have all the fancy kits. They only had the protocols in Sambrock and Maniatis Molecular cloning and they minipreped just fine:) But those protocols result in a quality not appropriate for sequencing (I had bad experience with that). But for most of other applications, you save lots of money with them. Just to put it in numbers: 250 columns are 125€ while the kit with 250 columns + some simple buffers is 243€.
@phage434: thanks for the Qiagen buffers. Our technician will be blissful tomorrow:)
Could be, but I always wonder: is it really worth the effort to make it yourself? You have to prepare it, it takes time too.
And if you need to make things fresh before using it.. I dont know.. it seems a lot easier to just use the kit...
And there will always be a difference in preparing it, esp if different people prepare the buffers.
I do agree on buying new columns (just columns) if you still have enough buffer left, in stead of always buying complete new kits.
Yes, we had same problem before, always run out of columns and have leftover solutions for DNA and RNA kits. We have been using Enzymax RNA/DNA mini spin columns for the past 6 or 8 years and they are so cheap, $39/100 DNA columns and $59/100 RNA columns . Now, they are selling a very tiny column call micro spin column for RNA and DNA, you can isolate DNA from single colony and elute the DNA in 5ul ddH2O.
I buy mini-prep kit from a Korean company http://solgent.com/english/pro1_2_1_01
Very cheap, easy protocol, and gives me clean product with high yield.
They do sell column and reagent separably
We did use successfully the columns from http://www.nbsbio.co.uk/catalogsearch/result/?q=spin+column (£27/100).
Buffers P1 and P2 where from http://openwetware.org/wiki/Qiagen_Buffers
- 50 mM Tris-HCl pH 8.0
- 10 mM EDTA
- 100 μg/ml RNaseA (choose from Sigma the one DNases-free or boil 15 min the one non-DNase-free (as from Sigma specification eventually)(RNasA is stable also at 100 C apparently)
- 200 mM NaOH
- 1% SDS
Buffer N3 did not work for us.
We found that the binding agent was alchool and likely Guanidinium chloride is more a denaturant for DNases (in fact buffer PE still allows binding of DNA to the silica membrane and it contains Tris and ethanol only).
0.9 M potassium acetate is needed to precipitate SDS and avoid contamination with genomic DNA.
Likely 0.9 potassium acetate plus ethanol or isopropanol should work well (and EDTA 10 mM to inhibit DNAses then lost through the column).
Anyway this mix should be tried on a small scale as I am not aware if it could produce any reaction (flammable or toxic).
The addition of Guanidinium chloride may work also better bu is to be checked for any unexpected chemical reaction eventually (toxic or flamable?)