This works for any size oligo that I have tried, the smallest being under=
10 bases and=20
the largest that I have attempted being around 170 bases. What varies is =
the=20
concentration of the gel mixture. I have used both the acrylamide and bis=
-acrylamide=20
powders, and the standard premixed solutions available and found no diffe=
rence. The=20
premixed solution is much easier to work with of course, but I know some =
people like to=20
make their own solutions. For anything up to 50-60 bases I use 12% PAGE w=
ith 7M urea,=20
for 60-90 bases I use 10%, for 90-120 I use 9%, for the 170-mers I used =
8%. My plates=20
are about 25 cm square, my spacers 1.5 mm, my wells about 3 cm, I run the=
gel at 400-430=20
volts, with a fan close to the plates for cooling (I have gel boxes more =
than 10 years=20
old with no cooling, but I=92m sure the more modern ones that are water c=
ooled would work=20
as well). I put a whole 40 nM synthesis in one well, for 0.2 uM I divide =
the amount over=20
3 wells, and for 1.0 uM I divide over 9 wells. Running time depends on th=
e length of=20
course, 50 minutes for a 20-mer, 80 minutes for a 50-mer, about 90 minute=
s for 100-mer,=20
and so on. The time is not proportional to the length exactly because as =
you are=20
decreasing the gel concentration, it will run faster even though it=92s l=
onger. I=92m sure=20
anyone who has done gels can see this.
My protocol is simpler because I have not found the need for any addition=
al manipulation=20
after deprotection. I evaporate the ammonium hydroxide by speed vac-ing o=
r DMA-MATE=20
block, then resuspend the DNA in a 50:50 mixture of water and loading buf=
fer. Typically,=20
I add 60 uL water and 60 uL loading buffer to a 40 nM synthesis and load =
the whole 120=20
uL in one well. (the loading buffer is 40% glycerol in TRIS buffer, no ED=
TA). I do not=20
add the blue dyes to the samples, I run them in much smaller wells on eac=
h side of the=20
plate.
I use UV shadowing to cut out the bands, cut the gel into pieces approx. =
1 mm square,=20
divide the pieces into two 1.5 cc microcentrifuge tubes, cover them to th=
e top of the=20
tubes with TRIS buffer, and incubate at least overnight in a 55 degree C =
dry heating=20
block. Vortexing the tubes occasionally I think improves the recovery, bu=
t I have no=20
figures on it, just my feeling. The next day I remove the buffer, wash wi=
th fresh buffer=20
and combine the 2 supernatants, and desalt on a Sep-Pack column (C18, Wa=
ters). The=20
final elution from the Sep-Pack is in 60% methanol/water, I then speed va=
c to dryness,=20
resuspend in 100 uL water and read the OD to quantitate.=20
This takes a day and a half, but I have been using this procedure in my f=
acility for=20
more than 10 years with very few problems and have been asked to gel puri=
fy DNA made in=20
other facilities, presumably because of the good results. The recovery is=
not 100%, and=20
I=92m sure I can improve it, but at the expense of additional time and ma=
npower, neither=20
of which I have enough. But for someone doing this once in a while only, =
vortexing the=20
tubes with the gel pieces helps, also removing the first supernatant afte=
r a few hours=20
and re-incubating with fresh buffer for a few more hours increases the re=
covery=20
(combining the 2, of course, before the desalting). Electroelution might =
work better,=20
but I have no experience with it.
I hope this helps, for more details just ask.