Southern California Biotechnology Center (SCBC) at Miramar College: Amgen
Biotech Experience (ABE) Program
Standard Operating Procedure (SOP): December, 2015
Title: Preparing 10X Restriction buffer for Amgen Labs 2 and 2a
Materials/Supplies:
pH Meter
Scale
Spatula
Weigh paper or trough
Graduated cylinders
Beakers
Storage bottles
Tris base
Magnesium Chloride (MgCl2)
Sodium Chloride (NaCl)
Dithiothreitol (DTT)
Hydrochloric Acid (HCl), Concentrated
dH2O
Prepare the following reagents or use ones that have been prepared by a reputable source
to make the 10x Compromise buffer.
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 (m.w. 121.10) in 80
ml deionized or distilled water. Check the pH with pH meter. The solution should
be basic.
2. Adjust to pH 8.0 by slowly adding concentrated HCl. Monitor the pH with pH
meter.
3. Transfer solution to 100 ml graduated cylinder and add deionized or distilled
water to a final volume of 100 ml.
4. Transfer solution to 100 ml or larger storage bottle. Label bottle with contents,
pH, date prepared and your initials. Store at room temperature.
1 M Magnesium Chloride (MgCl2)
Makes 100 ml. Store at room temperature indefinitely
1. In a 100 ml beaker or larger, dissolve 20.3 grams of MgCl2 (6-hydrate, m.w.
203.30) in 80 ml deionized or distilled water.
2. Transfer solution to 100 ml graduated cylinder and add deionized or distilled
water to a final volume of 100 ml.
3. Transfer solution to 100 ml or larger storage bottle. Label bottle with contents,
date prepared and your initials. Store at room temperature.
5 M Sodium Chloride (NaCl)
Makes 100 ml. Store at room temperature indefinitely.
1. In a 100 ml beaker or larger, dissolve 29.2 grams of NaCl (m.w. 58.44) in 80 ml
deionized or distilled water.
2. Transfer solution to 100 ml graduated cylinder and add deionized or distilled
water to a final volume of 100 ml.
3. Transfer solution to 100 ml or larger storage bottle. Label bottle with contents,
date prepared and your initials. Store at room temperature.
1 M Dithiothreitol (DTT)
Makes 10 ml. Store at -20 ºC indefinitely.
1. In a 10 ml beaker or larger, dissolve 1.5 grams of DTT (m.w. 154.25) in 8 ml
deionized or distilled water.
2. Transfer solution to 10 ml graduated cylinder and add deionized or distilled water
to a final volume of 10 ml.
3. Contents, date prepared and your initials. Place tubes in storage box. Label box
with contents and store at -20 ºC.
Prepare 100 ml of 10X Restriction buffer as below: (this is the STOCK solution)
10 mL of 1 M Tris pH 8.0
10 mL of 1 M MgCl2
20 mL of 5 M NaCl
1 mL of 1 M DTT
59 mL of dH2O
Store at 4oC
Prepare 200 ml 2.5X Restriction buffer as below: (this is the WORKING solution)
1. Mix 50 ml of 10X Restriction buffer with 150 ml of deionized or distilled water.
2. Aliquot 1.4 ml of 2.5X restriction buffer into 1.5 ml microfuge tubes labeled
“2.5xB”
3. Store tubes at 4 ºC indefinitely.