Tri-Nerd finds a summer job

Here's my job! Takes about seven and half hours straight to do one of these puppies...Ouch

F2 – Isoprostane Assay

THAW, CENTRIFUGE PLASMA SAMPLES BEFORE STARTING

Label plasma tubes with initial, plasma number
2ml Di H20
20 ml internal std. (d4 8-iso PGF2a, found in freezer) with Hamilton syringe; clean with ethanol following use, touch syringe to side of tube

Add sample to tubes
RECORD VOLUME USED!
Plasma: pipette as much as possible from thawed tubes and record volume
Urine: 200 ml
Vortex

Adjust sample pH to 3 using 1N HCL
Using a pipette top touch small amount of sample to pH paper
Vortex

Put C18 Sep-Pak into place
Syringes, label with sample number and initial
KEEP AND LABEL SYRINGE STOPPERS
Waste tubes, label with sample number and initial then put into corresponding place with C18 Sep-Pak
Precondition with 5ml methanol, then 7ml pH3 H20

Decant waste into waste beaker
Vortex samples before adding
Add samples, do not push through too rapidly
Label new, clean tubes with sample number and initial

Wash samples
10ml pH3 H20
10ml heptane

Add new, clean labeled tubes
Decant waste into waste beaker
Elute sample into new tubes with 10ml 1:1 ethyl acetate:heptane
Remove tubes, replace with waste tubes

Remove C18 Sep-Pak and replace with silica Sep-Pak (black tops)
Pre-rinse with 5ml ethyl acetate

9/10. Add small scoop of sodium sulfate and vortex lightly
Pour into silica Sep-Pak, *Do not to pour in sodium sulfate*
Push through slowly
Wash samples
5ml ethyl acetate
Label small tubes and tops with sample number and initial

Add new small tubes and pour off waste into waste beaker
Elute sample into small tubes with 5 ml 1:1 ethyl acetate:methanol

Dry samples under N2 bath at 37 degrees for *25min* or until evaporated

Esterify samples
40 ml PFBB
20 ml DIPE
Vortex
Place at 37 degree (wet incubator) for *20min*

Dry samples under N2 bath (approx 5min)

Reconstitute sample
50ml 3:2 methanol:chloroform
Make new reconstitution of 3:2 methanol:chloroform each time

TLC Purification
Make sure that the TLC plates have been prepared fresh daily
Draw line across TLC plate 13cm from origin
Spot 5ml TLC std. (PGF2a TLC std) to separate TLC plate
Spot samples - 50ml sample
Dry samples and place samples in TLC tank, allowing solvent to move to the 13cm mark before removing from tank, dry plates
Mark centrifuge tubes with sample number and initial
Spray std. with phosphomolybdic acid, place std. on hot plate until dark band appears (1-2min) then mark 1cm above and 1cm below mark
Pair samples with mark from std. plate, scrape samples onto filter plate and pour into marked centrifuge tubes
Add 1.25ml ethyl acetate,
Vortex

Centrifuge at max rpm for *2min*
Label new centrifuge tubes with sample number and initial

Use 1ml pipette to decant ethyl acetate into new centrifuge tubes, *make sure not to decant any silica*

Dry samples under N2 bath at 37 degrees (approx 15min)

Add 8ml DMF, 20ml BSTFA (located in brown, sealed containers)
Vortex, place at 37 degrees C for *5min*

Dry under N2
Reconstitute with 15ml undecane
Label and prepare GC tubes
Pipette out samples, cap samples