General Protocols¶
0.1
Common Buffers¶
Cell Culture¶
1x PBS¶
To 800ml ddH2O add:
8g NaCl 0.2g KCl 1.44g Na2HPO4 (sodium phosphate dibasic) 0.24g KH2PO4 (monopotassium phosphate)
pH to 7.4, and bring up to 1L with ddH2O. Autoclave to sterilize.
10x PBS¶
To 800ml ddH2O add:
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pH to 7.4, and bring up to 1L with ddH2O. Autoclave to sterilize.
50x TAE¶
For each litre of solution:
242g Tris Base 57.1ml Glacial Acetic Acid 100ml 0.5M EDTA
Mix Tris with stir bar to dissolve in about 600ml of ddH2O. Add the EDTA and Acetic Acid. Bring final volume to 1L with ddH2O. Store at room temperature.
Note: Final (1x) working concentration:
0.04M Tris-Acetate 0.001M EDTA
Western Blotting¶
Lysis Buffer¶
| Reagent | Stock Solution | Volume |
|---|---|---|
| ddH_2O | – | 7.68 ml |
| Tris pH 8.0 | 20 mM | 200 ul |
| NaCl | 0.15 M | 750 ul |
| EDTA | 2 mM | 40 ul |
| NP40 | – | 100 ul |
| Glycerol | – | 1000 ul |
| Na3VO4 | 1 mM | 100 ul |
The above recipe is for 10ml. This buffer can be made in bulk and used as a wash buffer, or allotted and frozen for future use. Immediately before use add protease inhibitors at the manufacturers recommended concentration, and phosphatase inhibitors if required.
Laemmli’s Buffer, 4x
2.4 ml 1 M Tris pH 6.8 (Same as upper gel buffer) 0.8 g SDS stock 4 ml 100% glycerol 0.01% bromophenol blue. Final Concentration is .02% 2.8 ml ddH2O
Before use add 1/10th volume of β-mercaptoethanol
Laemmli’s Buffer, 6x
1.2g SDS (sodium dodecyl sulfate) 0.01% bromophenol blue 4.7ml glycerol 1.2ml Tris 0.5M pH6.8 2.1ml ddH2O
Before use add 1/8th volume of β-mercaptoethanol
4x Lower Gel Buffer
182g Tris-Base (1.5M) 1L dH2O
pH to 8.8; do not overshoot. Use glass pipettes to pH.
4x Upper Gel Buffer
30.28g Tris-HCl (0.5M) 500ml dH2O
pH to 6.8, do not overshoot. Use glass pipettes.
10x Running Buffer
30.4g Tri-HCl 144.2g Glycine 10g SDS
Dissolve in 1L dH2O. Dilute 1:10 in ddH2O for use as running buffer
10X Transfer Buffer (Towbin Buffer)
30.3g Tris-Base 144.15g Glycine 100ml 10% SDS (0.1%)
Bring upto 1L in ddH2O.
1X Transfer Buffer (Towbin Buffer)
100ml 10X buffer 200ml methanol 700ml ddH2O
10x TBS
302.5g Tris-Base 400g NaCl 18g KCl
Dilute to 5L in dH2O, pH to 7.5 with saturated HCl.
Wash Buffer (TBST)
to 1X TBS ADD:
- Standard
- 0.1% Tween-20
- Strong
- 0.1% Tween-20 + 0.1% NP-40
- Extra-Strong
- 0.1% Tween-20 + 1.5% NP-40
Note: Standard buffer is for most application. Strong/extra strong are only for antibodies with a high degree of non-specificity.
DNA Buffers
6X DNA Loading Buffer
6.7 ml of ddH2O 10 mg of bromophenol blue 10 mg of xylene cyanol FF (optional) 3.3ml glycerol
Notes:
Bromophenol Blue runs at ~300bp (should be added to avoid over-running of gels) Xylene cyanol FF runs at ~4000bp (optional dye) 60 mM EDTA can be added to inhibit DNA-altering enzymes (600ul of 1M EDTA; reduce water appropriately)