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:
- 80g NaCl
- 2.0g KCl
- 14.4g Na2HPO4 (sodium phosphate dibasic)
- 2.4g KH2PO4 (monopotassium phosphate)
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 |
ddH2O |
||
Tris, pH 8.0 |
||
NaCl |
||
EDTA |
||
NP40 |
||
Glycerol |
||
Na3VO4 |
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)