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Leucine
Incorporation Rate, Field Procedures
Byron Crump, July 29, 2003
Equipment
Designated
'Radioactive' Cooler
Field notebook (e.g. "Rite in the Rain")
Sharpie pen
Pencil
1 gallon "rad waste" ziplock bag taped to inside of
Rad. cooler
Small round styrofoam cooler (~10" tall and 5" in
diameter, the kind used for shipping glass bottles
of chemicals)
Blue-ice bag that will fit in cooler (frozen)
4 1L plastic sample bottles (acid washed and
sterile)
4 Thermoses (1 quart, stainless steel, wide
mouth)
Incubation vials: each consists of
20 ml scintillation
vial (HDPE) pre-sterilized, uncapped (e.g. Fisher
#03-337-23B)
Black open top cap
(Fisher #NC9652480)
22 mm Teflon/Silicon
septa (sterile) (Fisher #NC9706925)
4X10 rack for vials
25 ml plastic pipette and pipette bulb
20-200 ml pipette
1 Rack of 20-200 ul pipette tips (sterile)
5 cc syringe with needle
0.22 um 25 mm isopore filters (Millipore #GTBP02500)
filter forceps
Filtration rig for 25 mm diameter filters
designated 'radioactive'
7 ml scintillation vials (e.g., Fisher #033371)
squirt bottle for 5% trichloroacetic acid
gloves
Solutions
3H-leucine stock solution (~99 Ci/mmol; e.g.
ICN # 2003605)
10 uM 3H-leucine working solution diluted in sterile water
100% trichloroacetic acid solution (100% TCA e.g.,
Sigma 490-10)
5% trichloroacetic acid solution (5% TCA)
Ethylene glycol monoethyl ether ("Methyl
Cellusolve", Fisher #E1801)
Scintisafe 30% liquid scintillation cocktail
(Fisher #SX23-5)
Procedure
Preparation
Assemble and number the incubation vials
(cap, septa with teflon side down, vial)
Put 3H-Leucine working solution in sealed plastic
container in small styrofoam cooler with a frozen
blue-ice bag.
In
the field
Collect sample in 1L bottle and in
thermos.
Do all sample manipulation in the 'Radioactive'
cooler.
Load 5 cc syringe with 100% TCA.
Open 4 incubation vials per sample.
Pipette 5 ml of sample (from bottle) into each
vial.
Add 0.25 ul 100% TCA to the first "T0" vial with syringe and swirl.
Put on gloves.
Add 25 ml 3H-leucine to each vial with new sterile tip (final concentration 50 nM Leucine, or approximately 25 uCi per vial)
Eject tip into Rad solid waste bag
Close vials carefully, and swirl to mix.
Submerge vials in thermos and close thermos.
Incubate 1 hour.
Remove vials from thermos (its OK to pour the
liquid out of the thermos)
Add 0.25 ml 100% TCA to the three "T1" vials
through the septa with the syringe and needle, and
shake.
Return vials to the rack.
ool and store in refrigerator
In
the lab
Filter samples through 25 um 0.22 mm Millipore filters
Rinse vials with ice-cold 5% TCA and pour through
filters
Turn off vacuum and cover filters with 5 ml
ice-cold 5% TCA
Incubate for 5 minutes
Rinse filter 2 times with 5 ml ice-cold 5% TCA
Transfer filters to 7 ml scintillation vials
Add 1 ml methyl cellusolve and incubate until
filters are transparent
Add 6 ml Scintisafe 30% scintillation cocktail
Count radioactivity in scintillation
counter
Modified
from Kirchman, D. et al. 1985. Leucine
incorporation and its potential as a measure of
protein synthesis by bacteria in natural aquatic
systems. Appl. Environ. Microbiol.
49:599-607
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