General buffers |
| 1 X PBS: |
| To make 1 L |
| 8 g NaCl (mw: 58.4 g/mol) |
| 200 mg KCl (mw: 74.551 g/mol) |
| 1.44 g Na2HPO4 (mw: 141.96 g/mol) |
| 245 mg KH2PO4 (mw: 136.086 g/mol) |
| Add distilled water to make volume 1 L |
| Adjust pH to 7.4 |
| 0.5 M EDTA, pH 8: |
| Add 400 mL distilled water |
| Add 93.05 g Na2•EDTA•2H2O (disodium dyhydrate) |
| 9 g solid NaOH |
| Adjust the pH with with 10 N NaOH |
| Add distilled water to make volume 500 mL |
| 10 M NaOH (= 10 N NaOH): |
| To make 500 mL |
| 200 g NaOH in a final volume of 500 mL distilled water |
| 5 M NaCl: |
| To make 1 L |
| 292.2 g NaCl in a final volume of 1 L distilled water |
| 1 M HEPES stocks: |
| To make 1 L |
| 238.3 g HEPES (free acid) |
| 800 mL of ddH2O |
| Adjust the pH to the desired value with 10 N NaOH |
|
Add distilled water to make volume 1 L |
For SDS PAGE |
| 10x Tris-glycine running buffer: |
| To make 4 L |
| 121.1 g Tris base |
| 576 g glycine |
| 200 mL 20% SDS |
| Add distilled water to make volume 4 L |
| 1 x Running buffer: |
| To make 1 L |
| 100 mL 10x running buffer |
| 900 ml distilled water |
| For making main separating gels: |
| 1.5 M Tris, pH 8.8 : room temperature |
| To make 1 L |
| 800 mL distilled water |
| 181.65 g Tris base |
| Adjust the pH to 8.8 with concentrated HCl |
| Add distilled water to make volume 1 L |
| For making stacking gels: |
| 1.5 M Tris, pH 6.8 |
| 181.65 g Tris base |
| Adjust the pH to 6.8 with concentrated HCl |
| Add distilled water to make volume 1 L |
For western blot |
| 10x Transfer buffer: |
| To make 4 L |
| 121.1 g Tris base |
| 576 g glycine |
| Bring up the volume to 4 L with ddH2O |
| 1x Transfer buffer: 4 C temperature |
| To make 1 L |
| 700 mL cold ddH2O |
| 100 mL 10x Transfer buffer |
| 200 mL methanol |
| Blocking buffer: 1-3% usually works |
| to make 100 ml |
| 1-3 grams of skim milk powder |
| add 1X PBS to make volume 100 ml |
| 20x TBS: |
| To make 4 L |
| 193.6 g Tris base |
| 640 g NaCl |
| Add distilled water to make volume 3.2 L |
| Adjust pH to 7.6 by adding HCl |
| Add distilled water to make volume 4 L |
| 20X TBST: |
| To make 100 mL |
| 2 mL Tween-20 |
| 100 mL of 20x TBS |
| 1X TBST: also works using 1X PBS |
| To make 1 L |
| Add 500 ul Tween-20 |
| Add 1X PBS to make volume to 1 L |
| RIPA buffer: |
| To make 10 ml |
| NaCl (1 M) 1.5 ml to make cocentration 150 mM |
| Nonidet P-40 (1%) 0.1 ml in 10 ml |
| Sodium deoxycholate (DOC) (0.5%) 0.05 ml |
| SDS (0.1%) 0.01 mL |
| Tris (50 mM, pH 7.4) 5 ml to make concentration 25 mM |
| Add distilled water to make total volume 10ml |
| 6X Loading buffer |
| 0.3 M Tris-HCl |
| 0.6 M DTT |
| 10% SDS |
| 0.06% Bromophenol blue |
| 30% Glycerol |