DRL Lab 1-3
Iron Gel (Fe(III)) Solution
Use iron contaminated beaker, stir bar, centrifuge bottles, and disposable pipettes.
A. Dissolve 108g Ferric Chloride (FeCl
3
* 6 H
2
O)
1.
Place 800 ml of Milli-Q H
2
O in a 2L beaker and place on a stir plate with a large stir bar.
2.
Weigh out 108g of Fe Chloride.
3.
Add Fe Chloride to the water.
4.
Once the Fe Chloride is fully dissolved remove approximately 50ml of this solution and set aside.
B. pH the Fe Chloride Solution to 7.0
5.
Calibrate your pH meter and place the electrode into the large beaker of Fe
Chloride.
6.
pH the solution to 5.0 using 10N NaOH. a.
It should take a lot of NaOH, but do not add the sodium hydroxide too quickly. b.
It is best if you add it a few milliliters at a time with a pasteur pipette. c.
The solution is going to look very clumpy d.
Rinse the electrode often with Milli-Q water to maintain accurate readings.
7.
When the pH reaches about 5.0 the sodium hydroxide should finally get mixed in and the solution will become very thick. a.
Make sure the solution is still mixing b.
Rinse the electrode often with Milli-Q water to maintain accurate readings.
8.
Switch to 1N or 2N NaOH and continue to slowly add until you reach pH 7.0. a.
If pH goes over 7.00, use set aside pure Fe Chloride solution to bring it back down. i.
Use a pasteur pipette to add dropwise, as it does not take much.
9.
Once you have reached pH 7.0 allow the solution to sit and spin for 30 minutes. a.
After 30 minutes recheck the pH of your solution i.
If necessary adjust accordingly so it is at pH 7.0. ii.
It is not unusual for it to have dropped below 6.5.
C. Wash FeGel with Milli-Q Water by Centrifugation
10.
Distribute your solution evenly into six 500ml centrifuge bottles with lids. a.
Check for gaskets in each lid.
11.
Add milli-Q water to each bottle to bring up the volume of the solution to approximately 2/3 the volume of the vessel.
12.
Balance all the bottles by weighing each bottle on a scale and add Milli-Q water until each weighs the same. a.
Weights should all be within 1/10 th
of a gram. b.
Don’t forget to weigh the lid with the bottle!
13.
Seal tightly and centrifuge them at 5000 rpm for 15 minutes.
14.
Decant supernatant from each bottle.

DRL Lab 2-3
15.
Scrap the gel from the sides and add milli-Q water to resuspend it. a.
The best way to do this is to fill the bottle 2/3 of the way full, put the cover on, and shake vigorously until mixed.
16.
At this point you can stop and store the gel in Milli-Q water at 4ºC overnight.
17.
Repeat steps 12-16 to wash the solution 4-6 times until the supernatant is a yellow/brown color. a.
The gel is complete when the water left in the bottle after being centrifuged is no longer clear but yellow/brown.
18.
When you are confident the gel is finished, scrape all of it into one bottle, fill 2/3 with Milli-Q and store in 4ºC with a stir bar (Fe contaminated).
19.
Check the total Fe(III) concentration before using this.
D. Determine total [Fe (III)] in solution
This step can be done either just after the above or at any time thereafter.
1.
Shake bottle vigorously and place on stir plate. a.
Gel should be stirring well (with the little whirlpool in the middle) for at least 15 minutes before pipetting. b.
If the bottle cannot be made to stir well, shake vigorously for 3-5 minutes by hand and pipet quickly.
Hydroxylamine Digest of Fe Gel - First Dilution (1:50)
2.
Make 6.25N hydroxylamine in small beaker or 15mL tube by adding:
0.43g hydroxylamine per 1mL of Milli-Q H
2
O and mix well.
3.
Cut the end off a yellow pipette tip to extract the iron gel
4.
In a scintillation vial, mix together: a.
100µl Fe gel b.
4.7mL of 0.5N HCl c.
200µl fresh 6.25N hydroxylamine
5.
Close vial and mix.
6.
Allow mixture to digest overnight in the dark.
Second Dilution (1:50)
7.
In a 2 nd
scintillation vial add: a.
100µl of digestion mix b.
4.9mL 0.5N HCl
Measurement of Fe2 Standard Curve
8.
In a 3 rd
scintillation vial, add: a.
100µl of Second Dilution mix b.
4.9mL Ferrozine solution (found in 4 o
C walk-in).
9.
Mix well and pour a portion of the sample into a cuvette
10.
Ferrozine solution is used as a blank with 0mM Fe for measurement.
11.
Measure absorption at 562nm against the standard curve for Fe saved as FeTrev.
12.
Multiply the mM/L by 2500 to account for dilutions made (50 x 50).
13.
Final concentration of stock solution is ideally between 1-1.2M for media
E. Using stock solutions of Fe gel in media
1.
Calculate the amount of Fe gel needed to make 1L media with a final concentration of ~100-150 mM Fe.

DRL Lab 3-3
Desired Final Concentration (100mM) = mL of Fe gel to add to 1L media
Actual Concentration (M/L)
Example Calculations for sample X:
1) @ 562nm; concentration = 0.440 mM/L
2) Multiply by dilution # (50 x 50): 0.440mM/L x 2500 = 1100.0 mM/L
3) 1.1M/L = Actual concentration; Desired concentration = 100mM
4) V
1
C
1
= V
2
C
2
(1.1M/L)(X) = (0.100M)(1000mL) ie: 100mM/1.1 = 90.9 ie: Add 91mL of 1.1M Fe gel to make 1L of media with a final concentration of 100mM.