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Recipes, Buffers









EDTA PBST TAE Trypsin
ELISA coating Pepsin TBE TTE
Laemmli Sample Proteinase TBS TTE (Tris/Taps/EDTA)
Modified RIPA (RBC) Lysis TBST X-gal Staining Solution
NP-40 Cell Lysis RIPA TE  
PBS Sodium Citrate Tris-EDTA  






EDTA
(1mM EDTA, 0.05% Tween 20, pH 8.0):
EDTA (Sigma, Cat# E-5134) ----------- 0.37 g
Distilled water -------------------------- 1000 ml
Mix to dissolve. Adjust pH to 8.0 using 1N NaOH.
Then add 0.5 ml of Tween 20 and mix well.



ELISA coating buffer

To 900ml dH20, add:
5.3g of Na2CO3
4.2g of NaHCO3
1g sodium azide
Adjust pH to 9.6
Adjust volume to 1L with additional dH2O
Note: For coating step add 0.1-1ug/ml of coating antibody to buffer
and incubate at 4°C for 12-24 hours.



Laemmli Sample Buffer (2X)
4% SDS
20% glycerol
10% 2-mercaptoethanol (or DTT) (add immediately before use)
0.004% bromphenol blue
0.125M Tris-Cl, pH 6.8



Modified RIPA buffer
10 mM Tris, pH 7.4
100 mM NaCl
1 mM EDTA
1 mM EGTA
1 mM NaF
20 mM Na4P2O7
2 mM Na3VO4
1% Triton X-100
10% glycerol
0.1% SDS
0.5% deoxycholate
plus protease inhibitors: Aprotinin, leupeptin, pepstatin: 1ug/ml each
Add 1mM PMSF immediately before use.
See: C2605-70, Cell Extraction Buffer
P9070, Protease Inhibitor Cocktail



NP-40 Cell Lysis Buffer
50mM Tris-HCl pH 8.0
150mM NaCl
1% NP-40
plus protease inhibitors: Aprotinin, leupeptin, pepstatin: 1ug/ml each
Add 1mM PMSF immediately before use.
See: P9070, Protease Inhibitor Cocktail



PBS
(137 mM NaCl, 2.7 mM KCl, 10 mM Sodium Phosphate dibasic, 2mM Potassium Phosphate monobasic, pH 7.4)
For 1 liter of 1X Phosphate-buffered saline (1X PBS buffer):
- Dissolve in 800ml of distilled H2O:
8.0g NaCl
0.2g KCl
1.44g Na2HPO4
0.24g KH2PO4
- Adjust the pH to 7.4 with HCl or NaOH
- Add H2O to 1 liter.



PBST
(137 mM NaCl, 2.7 mM KCl, 10 mM Sodium Phosphate dibasic, 2mM Potassium Phosphate monobasic, 0.1% Tween-20, pH 7.4)
For 1 liter of 1X Phosphate-buffered saline with Tween-20 (1X PBST buffer) -Dissolve in 800ml of distilled H2O:
8.0g NaCl
0.2g KCl
1.44g Na2HPO4
0.24g KH2PO4
-Add 1ml Tween-20
-Adjust pH to 7.4 with HCl or NaOH
-Add H2O to 1 liter



Pepsin
Pepsin Stock Solution (1% in 10mM HCl):
Pepsin ------------------------------------- 100 mg
10mM HCl (pH 2.0) ---------------------- 10 ml
Mix to dissolve. Store at -20 ºC

Pepsin Working Solution (0.5% in 5mM HCl):
Pepsin Stock Solution (1%) ------------ 1 ml
Distilled water ---------------------------- 1 ml
Mix well.



Proteinase K Solution
(20 ug/ml in TE Buffer, pH 8.0):
TE Buffer (50mM Tris Base, 1mM EDTA, 0.5% Triton X-100, pH 8.0):
Tris Base -------------------------------- 6.10 g
EDTA ------------------------------------- 0.37 g

Triton X-100 ---------------------------- 5 ml
Distilled water ------------------------- 1000 ml
Mix to dissolve. Adjust pH 8.0 using concentrated HCl (10N HCl). Store at room temperature.




Red Blood Cell (RBC) Lysis buffer
To 800ml dH2O add:
8.3g ammonium chloride (NH4Cl)
1.0g potassium bicarbonate (KHCO3)
1.8ml of 5% EDTA
Filter sterilize through 0.2um filter.
Bring to final volume of 1000ml with dH2O.



RIPA buffer
50mM Tris, pH 8.0
150mM sodium chloride
0.1% SDS

0.5% Sodium deoxycholate
1% NP40
plus protease inhibitors: Aprotinin, leupeptin, pepstatin: 1ug/ml each
Add 1mM PMSF immediately before use.
See: R2031-75, RIPA Lysis Buffer
P9070 , Protease Inhibitor Cocktail



Sodium Citrate Buffer
(10mM Sodium Citrate, 0.05% Tween 20, pH 6.0):
Tri-sodium citrate (dihydrate) --------- 2.94 g
Distilled water --------------------------- 1000 ml
Mix to dissolve. Adjust pH to 6.0 with 1N HCl and then add 0.5 ml of Tween 20 and mix well.



50X TAE buffer
To 900ml dH2O, add:
242g Tris base
57.1ml Glacial Acetic Acid
18.6g EDTA
Adjust volume to 1L with additional dH2O



10X TBE buffer
To 800ml dH2O, add:
108g Tris base
55g Boric acid
9.3g EDTA
Adjust volume to 1L with additional dH2O



TBS
(50 mM Tris, 150 mM NaCl, pH 7.4)
For 1 liter of 1X Tris-buffered saline (1X TBS buffer)
-Dissolve in 800ml of distilled H2O:
8g Sodium chloride
0.2g Potassium Chloride
3g Tris base
-Adjust pH to 7.4 with HCl
-Add H2O to 1 liter.

For 1 liter of 10X Tris-buffered saline (10X TBS buffer)
-Dissolve in 800ml of distilled H2O:
80g Sodium chloride
2.0g Potassium Chloride
30g Tris base
-Adjust pH to 7.4 with HCl
-Add H2O to 1 liter.



TBST
(50 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.4)
For 1 liter of 1X Tris-buffered saline with Tween-20 (1X TBST buffer)
-Dissolve in 800ml of distilled H2O:
8g NaCl
0.2g KCl
3g Tris base
-Add 500ul Tween-20
-Adjust the pH to 7.4 with HCl
-Add distilled H2O to 1L

For 1 liter of 10X Tris-buffered saline with Tween-20 (10X TBST buffer)
-Dissolve in 800ml of distilled H2O:
80g NaCl
20g KCl
30g Tris base
-Add 500ul Tween-20
-Adjust the pH to 7.4 with HCl
-Add distilled H2O to 1L



1X TE buffer
To 990ml dH2O, add:
10ml  1M Tris-Hcl, pH 8.0
400ul  0.25M EDTA



Tris-EDTA Buffer
(10mM Tris Base, 1mM EDTA Solution, 0.05% Tween 20, pH 9.0):
Tris Base -------------------------------- 1.21 g
EDTA ------------------------------------- 0.37 g
Distilled water -------------------------- 1000 ml (100 ml to make 10x, 50 ml to make 20x)
Mix to dissolve.
pH is usually at 9.0 and then add 0.5 ml of Tween 20 and mix well.



Trypsin Solution:
Trypsin Stock Solution (0.5% in Distilled Water):
Trypsin -------------------------------- 50 mg
Distilled water ------------------------ 10 ml
Mix to dissolve. Store at -20 ºC.

Calcium Chloride Stock Solution (1%):
Calcium chloride --------------------- 0.1 g
Distilled water ----------------------- 10 ml
Mix well and store at 4 ºC.
Trypsin Working Solution (0.05%):

Trypsin stock solution (0.5%) ------------ 1 ml
Calcium chloride stock solution 1% ------ 1 ml
Distilled Water ----------------------------- 8 ml
Adjust pH to 7.8 with 1N N NaOH.

Add proteinase K to TE buffer until dissolved.
Then add glycerol and mix well. Aliquot and store at –20 ºC for 2-3 years.

Working Solution (1x, 20 ug/ml or 0.6 units/ml):
Proteinase K Stock Solution (20x) ------ 1 ml
TE Buffer, pH8.0 ------------------------- 19 ml
Mix well. This solution is stable for 6 month at 4 ºC.



1X TTE buffer
To 988ml dH2O, add:
10ml  1M Tris-Hcl, pH 8.0
400ul  0.25M EDTA
2ml Triton X-100



1X TTE (Tris/Taps/EDTA)
50mM Tris
50mM TAPS
2mM EDTA



X-gal Staining Solution (C. elegans)
(Modified from Dichek Lab, Gladstone/UCSF)
Materials:
Potassium Ferricyanide Crystalline
Potassium Ferricyanide Trihydrate
Magnesium Chloride
X-gal
DMSO
2% Formaldehyde, 0.2% Gluteraldehyde in 1X PBS
Microscope Slides
Pap pen (Electron Microscopy Sciences #22303)
Nuclear Fast Red (also called Kernechtrot)
Gel Mount (Biomeda, M01)

Solutions:
1) Solution A:
5mM Potassium Ferricyanide Crystalline
5mM Potassium Ferricyanide Trihydrate
2mM Magnesium Chloride in 1X PBS
(stored at 4°C, protected from light)
2) X-gal Stock Solution (40X): 40mg/ml in DMSO (100mg in 2.5ml DMSO)
(store at -20°C, protected from light)
3) Final X-gal Solution:
Dilute X-gal stock solution 1:40 in Solution A (first warm Solution A to 37°C to prevent precipitation of X-gal)