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Sample
collection for planktonic DNA
Byron Crump, Jan 2, 2003
Equipment:
Sterivex-GP
0.22 mm filter capsules (Millipore
#SVGP01050)
60cc or 120cc luer-lock syringes
3cc luer-lock syringes
0.2 mm pore size sterile syringe filter (e.g.,
Fisher #09-730-218 acrodisc)
Needles
Syringe tips (from 3 cc syringes) cut in half
(keep the closed end).
Solutions:
DNA
Extraction Buffer (DEB)
To prepare 50 ml DEB (all solutions
autoclaved):
|
volume
|
solution
|
final
concentration
|
|
5
ml
|
1.0
M Tris buffer (pH 8.0)
|
0.1
M
|
|
10
ml
|
0.5
M NaEDTA (pH 8.0)
|
0.1
M
|
|
5
ml
|
1.0
M Phosphate buffer (pH 8.0)
|
0.1
M
|
|
15
ml
|
5.0
M NaCl
|
1.5
M
|
|
10
ml
|
5%
CTAB (Hexadecyl trimethyl Ammonium
Bromide, Sigma #H5882)
|
0.5%
|
|
5
ml
|
H2O
|
(Note: If a precipitate forms in DNA extraction
buffer, just warm it to room temperature and it
will redissolve.)
Procedure:
Filter
sample:
Pull
plunger out of syringe (60cc or 120 cc).
Attach syringe to Sterivex filter.
Pour sample into syringe.
Insert plunger and push sample through Sterivex
filter.
Remove Sterivex filter.
Repeat. If filtering plankton, aim to get 500 ml
through the filter.
After
filtering:
Force
all water out of filter with air in syringe.
Shake last few drops out of filter forcefully.
Cap the outport of the Sterivex filter with tips
from 3 cc syringes cut in half. (Note: Close the
outports gently so the little caps don't split, but
make sure they are sealed tight)
Inject ~2 ml DEB into inport with a needle. Try not
to pierce the white filter inside. (You can put all
the DEB buffer in a 60 cc (or 10 cc) syringe fitted
with a syringe filter and just use it repeatedly
for each sample.)
Close inport with a 3 cc syringe.
Freeze ASAP, store at -80ºC
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