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

1. Mix Tris with stir bar to dissolve in about 600ml of ddH₂O.
2. Add the EDTA and Acetic Acid.
3. Bring final volume to 1L with ddH₂O.
4. 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	--	7.68ml
Tris, pH 8.0	20mM	200ul
NaCl	0.15M	750ul
EDTA	2mM	40ul
NP40	--	100ul
Glycerol	--	1000ul
Na3VO4	1mM	100ul

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 1/10^th 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 1/8^th 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:

1. Bromophenol Blue runs at ~300bp (should be added to avoid over-running of gels)
2. Xylene cyanol FF runs at ~4000bp (optional dye)
3. 60 mM EDTA can be added to inhibit DNA-altering enzymes (600ul of 1M EDTA; reduce water appropriately)