Southern California Biotechnology Center (SCBC) at Miramar College: Amgen Biotech
Experience (ABE) Program
Standard Operating Procedure (SOP): December, 2015
Title: Preparing 4M Ammonium sulfate (NH4)2SO4 and 10X Tris/EDTA (TE) for Amgen Lab 6
Materials/Supplies:
pH Meter
Scale
Spatula
Weigh paper or trough
Graduated cylinders
Beakers
Storage bottles
15 mL conical tubes
Tris base
Ammonium sulfate (NH4)2SO4
Ethylene Diamine Tetraacetic Acid (EDTA)
Hydrochloric Acid (HCl), Concentrated
Sodium Hydroxide (NaOH), Concentrated
dH2O
Procedures:
1 M Tris, pH 8.0
Makes 100 ml. Store at room temperature indefinitely
1. In a 100 ml beaker or larger, dissolve 12.1 grams of Tris base (MW 121.10) in 80 ml
deionized or distilled water. Check the pH with pH meter. The solution should be basic.
2. Adjust the pH by slowly adding concentrated HCl. Monitor the pH with pH meter.
3. In a 100 ml graduated cylinder, bring to a final volume of 100 ml with deionized or
distilled water.
4. Transfer solution to a 100 ml or larger storage bottle. Label bottle with contents, pH,
date prepared and your initials. Store at room temperature.
0.5 M Ethylene Diamine Tetraacetic Acid (EDTA)
Makes 100 ml. Store at room temperature.
1. Place a clean stir bar in a 200 ml beaker or larger.
2. Dissolve 18.6 g EDTA (MW = 372.24) in 80 ml deionized or distilled water.
3. Place beaker on a stir plate and stir.
4. Check the pH with pH meter.
5. Adjust the pH to 8.0 by slowly adding concentrated 6.0 M NaOH, continue to stir the
solution.
NOTE: Mix vigorously, EDTA will only dissolve after the pH has reached 8.0 or higher.
6. In a 100 ml graduated cylinder, bring to a final volume of 100 ml with deionized or
distilled water.
7. Transfer solution to 100 ml or larger storage bottle. Label bottle with contents, pH, date
prepared and your initials. Store at room temperature.
Use the above prepared solutions to prepare 10X TE Buffer (Elution buffer) for Amgen Lab
6:
Makes 100 ml. Store at room temperature.
1. Add 10 mL 1 M Tris, pH 8.0 and 2 ml 0.5 M EDTA to 88mL of dH2O.
2. Place in 100 ml bottle.
3. Use a piece of tape to label bottle with contents, pH, date prepared and who prepared by.
Prepare 1 liter of 1X TE Buffer (Elution buffer) from 10X TE buffer
1. Add 100 mL of 10X TE buffer to 900 ml of dH2O.
2. Place in 1 liter bottle.
3. Use a piece of tape to label bottle with contents, pH, date prepared and who prepared by.
4. Aliquot 15.0 mL of 1X TE buffer into 15 ml conical tubes pre-labeled “Elution buffer”
(EB).
5. Place tubes in rack for use in high schools.
4M Ammonium sulfate (NH4)2SO4 -- Binding Buffer (BB)
Makes 1.0 Liter. Store at room temperature.
1. In a 2 liter beaker, dissolve 528.6 g (NH4)2SO4 (MW = 132.14) in 500 ml deionized or
distilled water.
NOTE: This is a saturated solution and it will take some time for all the ammonium sulfate to
go into solution. Add the ammonium sulfate slowly.
2. In a 1000 ml graduated cylinder, bring to a final volume of 1000 ml with deionized or
distilled water.
3. Transfer solution to a 1 liter reagent. Label bottle with contents, date prepared and your
initials. Store at room temperature.
Prepare 2M Ammonium sulfate (NH4)2SO4 – Column Equilibration Buffer (CEB) and Wash
Buffer (WB)
Makes 500 ml, Store at room temperature.
1. Add 250.0 mL of 4M Ammonium sulfate (NH4)2SO4 to 250.0 mL of dH2O.
2. Transfer solution to 500 ml or larger storage bottle. Label bottle with contents, pH, date
prepared and who prepared by. Store at room temperature.
Prepare Amgen Lab 6 reagents as below for each kit:
1. Aliquot 15.0 mL of 4M Ammonium sulfate buffer into pre-labeled 15 ml conical tubes.
Tubes should be labeled “Binding buffer” (BB)
2. Aliquot 15.0 mL of 2M Ammonium sulfate buffer into pre-labeled 15 ml conical tubes.
Tubes should be labeled “Column Equilibration buffer” (CEB)
3. Aliquot 15.0 mL of 2M Ammonium sulfate buffer into pre-labeled 15 ml conical tubes.
Tubes should be labeled “Wash buffer” (WB)
4. Aliquot 15.0 mL of 1X TE buffer into pre-labeled 15 ml conical tubes. Tubes should be
labeled “Elution buffer” (EB)
5. Place tubes in rack for use in high schools.