Genomic Diversity Laboratory

Department of Ecology and Evolutionary Biology

University of Michigan

Recipes for Stock Solutions

Bromophenol Blue Loading Dye (6X)*

*dark blue runs faster on the gel

Recipe 1:

For 100 mL:

60 g glycerol into a 100-mL graduated cylinder
12 mL 0.5 M EDTA, pH 8
Add 10 mg bromophenol blue
Bring up the volume to 100 mL with ddH2O

Recipe 2:

100 mg Bromophenol Blue

6 g of Ficoll (Type 400)
Fill up to 40 ml with dH2O

Dissolve well. It takes a while. An option is to shake at 37 degrees overnight. Store at

room temperature.

GelRed Gel: Use dark 1.5 ml tubes. Store stock solution at dark place and room temperature.
Loading Dye Mix
Mix:
10 l GelRed
1000 l Loading Dye (6X)
Vortex and centrifuge

Dilute in water Mix

Mix:
1 l GelRed
100 l ddH2O

Ladder:
1 l Ladder
1 l Loading Dye (6X)
8 H2O
TBE (Tris-borate-EDTA) (10X)
108 gm Tris Base (Trizma)
55 g boric acid
40 ml 0.5 M EDTA (pH 8.0)

Add the above to 800 mL dH2O. Stir until dissolved. Adjust to 1 L with additional dH2O.
Store at room temperature.

0.5 M EDTA (pH 8.0)

Add:
186.1 g of disodium EDTA (Na2EDTA)
800 mL of dH2O
Adjust the pH to 8.0 with NaOH (~50 ml of NaOH)
Bring volume to 1 L with dH2O
Stir vigorously on a magnetic stirrer*
Sterilize by autoclaving
Store at room temperature

*The disodium salt of EDTA will not dissolve until the pH of the solution is adjusted to
8.0 by the addition of NaOH.

1 M Tris-HCl
Dissolve:
121. g Tris Base (TRIZMA)
700 ml dH2O
Add concentrated HCl to desired pH:
pH 7.4 : 70 mL
pH 8.0 : 42 mL
pH 9.0 : 8 mL
Fill up to volume 1 L with dH2O. Sterilize by autoclaving for 20 minutes on liquid
setting. Store at room temperature.

Tris-EDTA 100X
(1.0 M Tris-HCl, 0.1 M EDTA, pH 8.0)

157.6 g Tris-HCL
37.22 g EDTA
Bring the solution up to 1 Lt with H2O (before you must adjust the pH to 8 using NaOH)

If preparing only 250 ml

39.4 g Tris-HCL
9.03 g EDTA
 Bring the solution up to 250 ml with H2O (before you must adjust the pH to 8 using
NaOH)

EDTA 0.5 M
186.1 g EDTA
Bring the solution up to 1 Lt with H2O

If preparing only 250 ml

46.53 g EDTA
Bring the solution up to 250 ml with H2O (adjust pH to 8 with ~5 g NaOH)

DTT (Dithiothreitol) 400mg/ml

0.4 g in 1 ml H2O
If you will prepare the entire bottle of DTT (1 g) then you just add 2.5 ml of H2O

NaCl 5 M
292.2 g in 1 LT H2O

If preparing only 100 ml:

29.2 g in 100 ml H2O

5X Extraction Buffer 40 ml Stock

2 ml Tris + EDTA (100X)
7.6 ml EDTA (0.5 M)
0.4 ml NaCl (5 M)
30 ml ddH2O

SDS (Sodium dodecyl sulfate) 10%

Dissolve:

10 g of SDS in 80 mL of H2O, and then add H20 to 100 mL

Extraction Solution Recipe (1 RXN)

Water 520 ul
5X Extraction Buffer 160 ul
SDS 10% 80 ul
Prot. K 10 mg/mL 20 ul
DTT 400 mg/mL 20 ul
Total volume 800 ul