Molecular Weights of Some Common Atoms and Compounds
MW NaCl = 58.44
MW ß-mercaptoethanol = 78.13, density ß-mercaptoethanol = 1.1 g/ml
MW Tris Base = 121.1
MW Mg Acetate (MgAc) = 214.4
MW NaH2PO4 = 120
MW Na2HPO4 = 141.96
MW KCl = 74.56
MgSO4 = 120.37
Hints for Making Your Solutions
Always use the C1V1 = C2V2 equation when you are diluting stock solutions.
Always use the purest water available for your solutions. In this class it will be Milli-Q water. In other laboratories it may be de-ionized or distilled water.
DTT is expensive and it gradually evaporates from solutions in which it is dissolved. Therefore, add DTT to your solutions just before you use them. Do this only in the hood. Stock solutions of 0.5 M DTT are stored in the freezer. Remember to pH all Tris solutions with the calomel electrode. Also, do not place the electrode in your solution if it contains SDS. Adjust the pH before you add the SDS stock.
Necessary Stock Solutions
1 M Tris-HCl pH 7.6 at 4°C Note that this means you should pH the solution when it is at this temperature.
1 M MgAc
1 M NaCl
Recipes for ß-galactosidase Assay (Procedure 2.2)
Z Buffer (500 ml)
4.26 g Na2HPO4
2.40 g NaH2PO4
0.373 g KCl
0.06 g MgSO4
5 mM DTT **
Bring to volume of 500 ml with water. Adjust pH to 7
0.1 M Phosphate Buffer for ONPG pH 7.5 (100 ml)
| 0.2 M Na2HPO4•2H2O (MW = 178.05; use 35.61 g/l) |
41 ml |
| 0.2 M NaH2PO4•2H2O (MW = 156.01; use 31.21 g/l) |
9 ml |
| water |
50 ml |
Recipe for Extraction (Procedure 3.1)
Breaking Buffer (200 ml)
| 0.2 M Tris |
40 ml of 1 M Tris pH 7.6 stock |
| 0.2 M NaCl |
40 ml 1 M NaCl stock |
| 0.01 M MgAc |
2 ml 1 M MgAc stock |
| 5% glycerol |
10 ml 100% glycerol |
| 5 mM DTT |
2 ml 0.5 M DTT (add just before using) |
Bring to volume of 200 ml with water.
Recipes for Ion Exchange Chromatography (Procedure 3.4)
0.2 M NTM Buffer (1 liter)
| 0.2 M NaCl |
200 ml of 1 M NaCl stock |
| 0.01 M Tris |
10 ml of 1 M Tris pH 7.6 stock |
| 0.01 M MgAc |
10 ml 1 M MgAc stock |
| 5 mM DTT |
10 ml of 0.5M DTT (add just before using) |
Bring to volume of 1 liter with water.
0.5 M NTM Buffer (1 liter)
| 0.5 M NaCl |
500 ml of 1 M NaCl stock |
| 0.01 M Tris |
10 ml 1 M Tris pH 7.6 stock |
| 0.01 M MgAc |
10 ml 1 M MgAc stock |
| 5 mM DTT |
10 ml 0.5 M DTT (add just before using) |
Bring to volume of 1 liter with water.
0.2 M NTM with 10X Tris (1 liter)
| 0.2 M NaCl |
200 ml 1 M NaCl stock |
| 0.1 M Tris |
100 ml 1 M Tris pH 7.6 stock |
| 0.01 M MgAc |
10 ml 1 M MgAc stock |
| 5 mM DTT |
10 ml 0.5 M DTT (add just before using) |
Bring to volume of 1 liter with water.
Recipes for SDS-PAGE (Procedure 4.2)
Running Buffer (1X Laemmli Buffer) 0.025 M Tris pH 8.3 (1 liter)
3.03 g Tris base
14.4 g glycine
10 ml 10% SDS stock
Add most of water, adjust pH to 8.3, bring to volume of 1 liter.
Sample Buffer (4X Laemmli Buffer)
2.4 ml 1 M Tris pH 6.8 (upper gel buffer)
0.8 g SDS stock
4 ml 100% glycerol
0.01% bromophenol blue so .02%
1 ml ß-mercaptoethanol (electrophoresis grade)
2.8 ml water about
ßTV mls
Recipes for pouring your own gels (Not used in this manual)
Resolving Gel 8% acrylamide (15 ml)
3.8 ml 1.5 M Tris pH 8.8 (lower gel buffer)
4.0 ml 30 % acrylamide stock
7.0 ml water (degassed)
150 µl 10% SDS stock
150 µml APS stock
9 µl TEMED
Stacking Gel (8 ml)
1 ml 1 M Tris pH 6.8 (upper gel buffer)
1.3 ml 30 % acrylamide stock
5.5 ml water (degassed)
80 µl 10% SDS stock
80 µl APS stock
8 µl TEMED
Recipes for Gel Staining (Procedure 4.3)
Coomassie Blue Stain
400 ml methanol
100 ml acetic acid
2.5 grams Coomassie Brilliant Blue
500 ml water (add water last)
Filter with Whatman paper #1 -
Stain can be reused several times but staining will lose intensity over time.
Coomassie Blue Destaining Solutions
40% methanol + 10% acetic acid (v/v) (30 minutes)
To speed up the destain process, a small piece of foam rubber can be placed in the container with the gel.
For 1 liter:
400 ml methanol
100 ml acetic acid
500 ml water
Silver Staining Reagents
Follow Bio-Rad Kit directions
Recipes for Western Blotting (Procedure 4.4)
Transfer Buffer 25 mM Tris (1 liter)
3.03 g Tris base
14.4 g glycine
200 ml methanol
Bring to volume of 1 liter with water, store at 4°C. Do not add acid or base to adjust pH. The buffer will range from pH 8.1 to 8.4 depending on the quality of Tris, glycine and methanol.
Ponceau S Stock/Working Solution
2% Ponceau S
30% trichloroacetic acid
30% sulfosalicylic acid
For staining, dilute concentrated dye solution 1 in 10 with water to make working solution, store at room temp. This stock is stable for 1 year.
Phosphate Buffered Saline (PBS) (1 liter)
8 g NaCl
0.2 g KCl
1.44 g Na2HPO4
0.24 g KH2PO4
Dissolve in 800 ml water. Adjust pH to 7.2 with H3PO4. Adjust volume to 1 liter.
10% Sodium Azide Stock
2 g sodium azide
Dissolve in 20 ml water. Sodium azide is poisonous. Avoid inhalation of the powder and use caution when handling the liquid.
3%Blocking solution
3 grams of BSA (non-fat dry milk can be substituted and is cheaper)
ßTV 100 mls with PBS
Tris Buffered Saline (TBS) (1 liter)
8.0 g NaCl
0.2 g KCl
3.0 g Tris base
Dissolve in 800 ml water. Adjust pH to 8.0 with 1 M HCl. Adjust volume to 1 liter, store at room temp.
Nitro Blue Tetrazolium (NBT) Stock (10 ml)
0.5 g NBT
10 ml of 70% dimethylformamide (DMF)
Store at 4°C for 1 year.
BCIP (Bromo-chloro-indolyl-phosphate) Stock Solution (10 ml)
0.5 g BCIP
10 ml of 100% DMF
Store at 4°C for 1 year.
Alkaline Phosphatase Buffer
100 mM NaCl
5 mM MgCl2
100 mM Tris
Adjust pH to 9.5, store at 4°C for 1 year.
Alkaline Phosphatase Substrate Solution (NBT/BCIP)
10 ml alkaline phosphatase buffer
66 µl NBT Stock
33 µl BCIP Stock
Use within 1 hour. Wear gloves when using NBT and BCIP.
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