A2 .
50 ml
In a 100 ml graduated cylinder measure
50 ml of unspiked
WW
Estimated time – 15 minutes
Kney – Update 1/28/08
A1.
Prefiltered
Wastewater
WW
A4.
Measured
500 ml
WW
A5.
E-4M spike
Transfer 50 ul of E2 to the
500 mL WW.
1500 mL
Beaker
What is the new concentration?
A3.
#1
Blank
Label w/Group #
Transfer the 50 ml unspiked WW sample to a 125 ml
Erlenmeyer flask and Label
1

Estimated time - 1.5 hours
0.05 grams
#2
Control
NO GAC
Label w/Group #
1500 mL
Beaker (500ml)
Spiked WW
#3
0.05gm
Label w/Group #
B2 . Transfer 50 ml of the spiked WW sample to each of the remaining 6-125 ml
Erlenmeyer flasks
B4
Beaker of GAC
0.10 grams 0.25 grams
0.10gm w/Group #
Same
B1 . Using 1-gram weigh boats, measure out various amounts of GAC. Be sure not to mix up measured amounts.
#4
Label procedure for remaining 2
Erlenmeyer flasks
. Place all 7 Erlenmeyer flasks
B3 procedure for
0.75g and
. Transfer weighed amounts of GAC to each of properly labeled 125 ml
Erlenmeyer flasks
#5
Same
1.00g
0.25gm
Label w/Group # on shake table for 1 hour
2

Estimated time - 1.5 hours
C1.
Stock sterile 2X medium (50 ml Beaker)
C3 . Label microfuge tube
M-NY and set aside for the time being.
C2.
Transfer 1.5ml of sterile
2X medium (NO YEAST) to a sterile microfuge tube
C4.
Transfer 34.3ml of sterile
2X medium (NO YEAST) sterile
250 ml beaker
C6.
Swirl 35ml
2X-yeast/medium mixture for at least
Microfuge tube of active yeast
C5.
Transfer 700ul of active yeast to the 34.3ml sterile 2X medium in the 250ml beaker
45 seconds
(2XY medium)
C7 . Label 18 standard curve tubes and 40 sample tubes as identified in Phase 3, Sections B, C, D,
& E. At this time also label 58 tubes for Phase 4. Label the same as Phase 3 tubes.
C8 . Dispense 500 µl of sterile, deionized water to each standard curve tube and tubes B8A and
B8B with a 5 ml tip on a repeating pipetter with the dial set to #5.
Beaker of sterile DI water 1S through
8S
Standard Curve Tubes
500ul of DI water
DM
SO
1SA – 8SA and 1SAB –
8SB and DMSO A and B
B8A B8B
Batch Tubes
B8A and B8B
500ul of DI water
3

Continued
C9
. Dispense 500 µl of 2X yeast/medium to each standard curve tube and all batch tubes
EXCEPT batch tubes B8A and B8B with a 5 ml tip on a repeating pipetter with the dial set to #5. (100X and 10X batch tubes included)
Beaker of 2X
Yeast/medium
(2XY medium) 1S through
8S DM
SO
B1 through
B7
Standard Curve Tubes
500ul of 2XY medium
1SA – 8SA and 1SAB –
8SB and DMSO A and B
All Batch Tubes
500ul of 2XY medium
Except B8A and B8B
100X dilution and 10X dilutions are included
C10 . Dispense 1 ul of E2 standard ( i.e., estradiol source standards in DMSO provided by instructor ) into corresponding standard curve tube now filled with 500ul of DI water and 500 ul 2XY medium.
1 2
Estradiol source standards
1µl from each tube with estradiol concentration into each standard curve tube
1.00 E-6M 5.00E-7M Tubes 2.50E-7M – 7.80E-9M
DM
SO
DMSO
Standard curve tubes
1 ul 1 ul Standard curve tubes filled with 500ul DI water and
1 ul
DM
SO
1 2
500ul 2X medium
1SA & 1SB 2SA & 2SB 3SA & 3SB through 8SA & 8SB DMSO A & B
1.00 E-9M 5.00E-10M Tubes 2.50E-10M – 7.80E-12M DMSO
4

Continued
C11. Transfer 500ul of the appropriate batch sample identified in Phase 3, Section C from the
Erlenmeyer flasks to microfuge tubes.
#1
Blank
Label w/Group #
500ul
B1
#2
Control
NO GAC
Label w/Group #
500ul
B2
#3
0.05gm
Label w/Group #
500ul
B3
#4
0.10gm
Label w/Group #
500ul
B4 etc .
…….. etc.
……..
Beaker of sterile DI water
Add 500ul of appropriate batch sample for microfuge tubes B1A – B7A and B1B – B7B. These are now 2X dilutions! Do you understand WHY?
C12. Transfer 10ul of the appropriate batch sample identified in Phase 3, Section D from the Erlenmeyer flasks to microfuge tubes and 490ul of DI water to each of the 100X dilution batch tubes.
490ul
#2
Control
NO GAC
Label w/Group #
10ul
B2
#3
0.05gm
Label w/Group #
10ul
B3
#4
0.10gm
Label w/Group #
10ul
B4 etc.
…….. etc.
……..
Add 10ul of appropriate batch sample for microfuge tubes B2A – B7A and
B2B – B7B and 490ul of DI water. These are now 100X dilutions.
5

Continued
C13. Transfer 100ul of the appropriate batch sample identified in Phase 3, Section E from the
Erlenmeyer flasks to microfuge tubes and 400ul of DI water to each of the 10X dilution batch tubes.
Beaker of sterile DI water
400ul
#2
Control
NO GAC
Label w/Group #
100ul
B2
#3
0.05gm
Label w/Group #
100ul
B3
#4
0.10gm
Label w/Group #
100ul
B4
Add 100ul of appropriate batch sample for microfuge tubes B2A – B7A and
B2B – B7B and 400ul of DI water. These are now 10X dilutions. etc.
…….. etc.
……..
C14. Label all microfuge racks as stated in Phase 3, Section B, Part 1. Place both racks w/tubes in 30C incubator identified by instructor for 48 hours.
6

Done in 2 Stages
A. Prepare lyse (i.e., Z buffer) with substrate (i.e., ONPG and DTT)
250 ml clean beaker
Z buffer
A1.
55ml
B. Transfer 100µl from each Phase 3 tube to a new, clean tube labeled earlier. Next transfer 900ul lyse buffer to each of the 58 Phase 4 tubes
Each of 58 Phase 3 tubes
B6.
100µl
B7.
900µl
Do B6
A4.
55µl
A3.
2310µl
before B7.
DTT
T
Work quickly for B7 to keep minimum.
ONPG time from first to last tube to a
A new Phase 4 tube for each
Phase 3 tube.
C11.
400µl to each tube
C9 . 30 ml
Stop Buffer, Na
2
CO
3
B8.
Incubate at 35C for up to 3 hours
C10.
400µl
7

D. Optical Density reading
D11. Transfer about 1ml from each tube to a cuvette
Phase III tubes
Cuvette
Use the same cuvette for a series of Phase 4 tubes so long as each successive tube has a stronger yellow color than the previous one.
For example, with the standard curve, measure tube 8, then tube 7, then tube 6, to tube 1. Measure the two reps as separate runs.
Use The DMSO tube as the blank for the standard curve.
Use B8A and B8B as blanks for the wastewater samples.
The spectrophotometer should be set at a wavelength of 420nm.
8