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 70C for 5-10 minutes, and then put in a temp-block at 37C 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).
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