Gel purification of vector DNA - low yield - (Feb/23/2009 )

planktonica on Mar 4 2009, 01:13 PM said:

did you add enough QG buffer to your gel slab? It is the QG buffer that causes the DNA to bind to the column

perneseblue on Mar 4 2009, 02:19 PM said:

I add 5 times as much QG as my sample volume, as directed in the protocol. I really get low vector concentrations (around 10% recovery) and I am not sure if this is giving me problems with my ligation.

The instructions in the Qiagen gel extraction kit say to add 3X the QG buffer as the sample volume. It is the pcr purification kit that requires a 5X buffer addition. Most likely the buffer and isopropanol concentrations are way off. However, you only need a very small amount of vector for ligations so even if you have a bad yield, you should still have enough to get a ligation to work. I typically use 25ng vector in a ligation but have used as little as 10ng with success.

My PI told me to use the PCR purification protocol with the gel extraction because we are not actually extracting from gel. But I will try to use 3X instead and see what I get.

Thanks!

OK, really dumb question, but in which stages do you add the isopropanol and the Na acetate?

The Na acetate is added with the sample-GQ buffer mix. I don't add Isopropanol but the PE buffer contains Ethanol. I purified both, PCR product again and vector with the kit and got a lot of PCR and low concentration of vector again. However, it seems like 30 ng/uL is not that bad for transformation purposes.

And Swanny, I don't find your question dumb at all!

I always warm up the elution buffer (EB, water, etc) to 60C before elution and it helps me a great deal with the concentration.

ruiN on Mar 8 2009, 12:52 AM said:

For me, I warm it up to 50deg before elution. The heat helps to evaporates the ethanol in the previous reagent. That was what my colleague told me, though I am not too sure how the evaporation of the ethanol helps.

moorele on Feb 23 2009, 09:34 AM said:

hello
when you are gel isolating the bands, for how long you were exposing the vector DNA on the gel doc? It might so happen UV nicks the DNA when exposed so long , moreover you should try the preparative UV which has got longer wavelength than the shorter ones.
It may also happen that the Ultra salt may be too old ( NaI) to dissolve the gel completely and the silica beads may be old to bind to the DNA. try to vortex the silica mixture with the DNA so that the Brownian motion continues and when you precipitate by centrifuging , you get a lots of silica attached to the DNA.
cheers

tuhin_genes on Mar 11 2009, 02:19 AM said:

Hey,

I have always hated the column extraction kit from Quiagen because the yeild is always so low! but they do a kit which uses a resin called QiaxII which i prefer and has always been ok for me... its a bit more fiddly but well worth it!