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 ERDC TN-DOER-C30
February 2003
4) Place supplemented MEM in the water bath for about 30 min.
5) Remove Ponasterone A standard and environmental samples (stored in 4-mL
amber vials) from the freezer. Vortex the standard and samples, and let vials
warm to room temperature.
6) Prepare serial dilutions of Ponasterone A standards in DMSO using positive
displacement pipettors as follows:
Tube
Well
Concentration
Concentration
Tube Number
M*
M
Make-Up
1
10,000
100
Ponasterone A Stock
2
3,000
30
120 L tube 1 + 280 L of DMSO
3
1,000
10
40 L tube 1 + 360 L of DMSO
4
,300
3
120 L tube 3 + 280 L of DMSO
5
,100
1
40 L tube 3 + 360 L of DMSO
6
,30
0.3
120 L tube 5 + 280 L of DMSO
7
,10
0.1
40 L tube 5 + 360 L of DMSO
* The Ponasterone A standards are made 100x.
7) Prepare serial dilutions of Ponasterone A standards in MEM as follows:
Well
Concentration
Tube Number
M
Make-Up
1
100
50 L Ponasterone A tube 1 + 5,000 L of MEM
2
30
10 L Ponasterone A tube 2 + 1,000 L of MEM
3
10
50 L Ponasterone A tube 3 + 5,000 L of MEM
4
3
10 L Ponasterone A tube 4 + 1,000 L of MEM
5
1
50 L Ponasterone A tube 5 + 5,000 L of MEM
6
0.3
10 L Ponasterone A tube 6 + 1,000 L of MEM
7
0.1
50 L Ponasterone A tube 7 + 5,000 L of MEM
8
0
10 L DMSO + 1,000 L of MEM
8) Pipet off the cell medium from the two 96-well plates with a multichannel pipettor.
The plates can be tilted at a 30-deg angle so that the sharp ends of the pipet tips
can reach the medium at the bottom of the wells.
9) Pipet 200 L of the Ponasterone A medium (prepared in step 7) into each well.
10) Dose each well in the first 3 columns with 1 L of iso-octane, and 1 L of the
environmental samples (in iso-octane) in triplicate wells over 4 Ponasterone A
concentrations using a positive displacement pipettor in Plate 1 as follows:
17
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