Michael L. Sullivan
mlsulliv at facstaff.wisc.edu
Mon Mar 11 21:21:40 EST 2002
At 12:29 AM 3/12/2002 +0000, you wrote:
Useful sounding protocols. Might I suggest a few options as well.
For getting PCR reactions ready for digestion, I like to simply spin it
through a G-50 column (like the ones used for sequencing clean up), then
use the flow through directly in the digestion.
As for gel purification, I find low melt agarose is an excellent choice for
a large amount of fragment (e.g. a vector) . Simple cut out the band of
interest (including as little gel as possible), add 3 ul water per mg of
gel, and melt. The resulting solution can be used directly in a ligation
reaction provided the DNA is a reasonable concentration.
When there's less DNA to work with, I like to use S&S NA45 paper, which I
think is probably no more difficult than your trough method below.
Another alternative (from Peter at Harvard) was suggested to me last week
following a post I made on this newsgoup. He told me he just cuts the band
out of regular agarose, puts it in a microfuge tube, freezes in liquid
nitrogen, the spins for 10-15 min at max speed. The supernatant contains
most of the DNA fragment which can then be precipitated. Peter said the
secret is to freeze in liquid nitrogen. No other way to freeze will
do. Sounds VERY simple. I'm planning on trying this one out soon.
>I received the following note from an individual...
> >I noticed that your protocol of cloning pcr products is simpler than
> >mine. I found it was necessary to clean-up my pcr products before
> >digestion. I either gel-purify, or phenol extract the DNA followed by
> >ethanol precipitation. I believe the clean-up step is basically to
> >remove the attached DNA polymerase, that otherwise will fill in or
> >modify the digested ends of the DNA. I wonder whether your method of
> >"PPT them down in NaCl and Isoprop" does the trick? It may selectively
> >precipitate down the DNA and leave the proteins (Taq or pfu DNA
> >polymerase) in the supernatant?
> >Could you email me your detailed protocol?
>In my haste, I neglected a few steps that I mistakenly presumed were
>"obvious". As this is my error, let me try to fill in some of the
>First Taq is inhibited by salt, so as soon as the PCR reaction is done I put
>in 1/10th vol of NaCl, and PPT with 2 vols isopropanol. (The best
>inactivator of Taq (which will end fill as it can survive phenol,
>Na-Acetate, and EtOH precipitation) is PEG, but PEG is only condusive to
>ligase and inhibits all other enzymes.) I resuspend the fragment and then
>digest as I would any other piece of DNA. I load the entire digestion on a
>gel and I do this for the vector as well.
>Once run into the gel, I cut large troughs directly in front of the desired
>band - about 3-4 X the thickness of the band and about 10% larger than the
>bands width. I do use EtBr to visualize and I conduct what I call a "one
>trough pull"... when ready to elute, I max the power supply on constant
>watts, or mA, and watch the band with a hand held UV light, with the room
>lights off. When the majority of the band is in the trough, harvest it only
>once. If the trough is cut right (learned by experience), about 90% of the
>band will be in the trough at the right time. One pull is all that is
>needed and I've never spent more than 3 mins pulling a trough. When the
>power is that high, you can literally watch the band "tumble" into the
>What is harvested (<800ul) is then dry butanol extracted down to about
>200ul. It is then extracted twice with phenol, 2x with phenol/clfm, and 2X
>with clfm. One ug of glycogen is added, 1/20th of Na-acetate, and 2-2.5
>vols of EtOH is then added to precipitate it down. The band and vector are
>then washed with 70% EtOH, air dried and resuspended in 55ul of TE of which
>5ul is used for a 1:100 OD-260 quantitation. The bands are checked by
>agarose gel electrophoresis and the ligation is peformed.
>Note: Oligos... we design our oligos with a 6-8 base pair overhang PAST
>then end of the restriction site. If you don't put an overhang on, you
>won't be able to cut your PCR fragments. For example, I design oligos with
>a 20 base pair match of the target sequence, add the 6 bases for the enzyme
>I want, then add another 6-8 bases that either match the target sequence
>(which is what I do) or use the suggestions in the NEB catalog. Without
>the overhangs, all you can do is blunt end ligate, or TA clone.
>We electroporate into home grown prepared bugs and plate on LB with whatever
>the selection marker requires.
> From an overnight PCR, I have often digested, "trough-ed", prepared the
>bands, and set up for ligations the same day. If there are sticky ends,
>I've electroporated as soon as an hour after a room temp incubation with
>ligase and/or the next day after an overnight 15'C incubation of the
>Hope this helps...
More information about the Methods