Home

Overview

People

Projects

Protocols

Data/Posters/Reports

Photos & Maps

Publications

Links

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


[Home] [Overview] [People] [Projects] [Protocols] [Data] [Photos & Maps] [Publications] [Links]