Qiaex problems

Colin Rasmussen colin at fungus.biochem.ualberta.ca
Wed Feb 21 18:49:18 EST 1996

t.barnett at path.utas.edu.au (Tim Barnett) wrote:

>I have had a lot of trouble with the quiex procedure as well.  I now extract DNA from LMP agarose using a simple phenol/chloroform procedure.
>1.  Excise band from LMP agarose
>2.  Melt for 5min at 70C
>3.  Cool for 30s at RT and add an equal volume of TE saturated phenol
>4.  Spin for 5min in a microfuge
>5.  Remove aqueous phase and re-extract with an equal volume of phenol
>6.  Extract with phenol/chloroform and ethanol ppt.
>Yeilds aren't great, but this method works well for me.

We usually excise the band from LMT agarose, melt the gel at 70ƒC for 5-10 minutes, and then put in a temp-block at 37ƒC to keep it melted.  The ligation is then set-up as follows:

4 ul 5 x BRL ligation buffer (contains PEG)
5 ul vector DNA (try for 200 ng in 5 ul gel)
5 ul insert DNA (try for 100 ng in 5 ul gel)
1 ul T4 ligase (Promega, 3U/ul)

Many of the perfectionists out there will say, but what is the vector/insert ratio. The answer is:

a) it varies depending on the size of the vector and insert.
b) who cares. I routinely ligate this way and I always get what I am looking for.

It is important apparently though what type of LMT agarose you use, and Sea-Plaque seems to work the best. I have had variable luck previously with BRL agarose.  Best part, is the yields are wonderful.

Colin (no affiliation with any of the suppliers listed above).

More information about the Methods mailing list