Buffer, reagent and solution recipes


Buffer recipe download

 

Buffer, reagent and solution recipes

 

 

SDS-PAGE Gel Loading Buffer (Laemmli Buffer)

62.5 mM Tris, pH = 6.8

2% SDS (w/v)

10% Glycerol

100 mM DTT

Bromophenol Blue (0.1%)

 

To prepare 10 ml of 4x:

2.5 ml of 1M Tris, pH 6.8

0.8 g SDS

4 ml glycerol (best to weigh out ~5g with tube on balance since pipetting glycerol is inaccurate)

0.64 g DTT

800 ul of 1% bromophenol blue solution

H20 to 10 ml

 

 

 

SDS-PAGE Gel Running Buffer

25 mM Tris

192 mM Glycine

0.1% SDS (w/v)

 

To prepare 1 Liter of 10x:

30.3 g Tris

144 g Glycine

10 g SDS

 

 

 

Semi Dry Transfer Buffer- 15% Methanol- for Western Blot Transfer (a.k.a Towbin Buffer)

25 mM Tris Base

192 mM Glycine

15% Methanol

 

To prepare 1 Liter of 10x:

3 g of Tris Base (MW: 121 g/mol)

14.4 g of Glycine (MW: 75 g/mol)

150 ml Methanol

Note:  Vary methanol as needed (0-20%)

 

 

 

 

Wet Transfer Buffer- 20% Methanol- for Western Blot Transfer

48 mM Tris

39 mM Glycine

1.3 mM SDS

 

To prepare 1 liter of 10x:

58g Tris base (MW 121)

29 g Glycine

4g SDS

 

To prepare 1 liter of 1x:

100 mL 10x wet transfer buffer

200 mL methanol

700 mL ddH2O

 

 

 

 

Mini Prep Alkaline Lysis Solutions

Alkaline Lysis Solution I

50 mM glucose

25 mM Tris-Cl (pH 8.0)

10 mM EDTA (pH 8.0)

(add Rnase I before use at 10ug/ml)

 

Alkaline Lysis Solution II

0.2 N NaOH

1% (w/v) SDS

 

Alkaline Lysis Solution III

3 M potassium acetate

glacial acetic acid (11.5 ml per 100 ml)

 

 

 

 

NETN

20 mM Tris pH 8.0

100 mM NaCl

0.5 mM EDTA

0.5% NP40

 

To prepare 500 mL:

10 mL 1 M Tris, pH 7.5

10 mL 5 M NaCl

0.5 mL 0.5 M EDTA

2.5 mL NP40

Bring to volume using double distilled water

 

 

 

 

Coomassie Blue

1 g R250 Coomasie Blue per Liter final

40% Methanol

10% Glacial Acetic Acid

 

For 1 Liter:

400ml Methanol

100ml Glacial Acetic Acid

1g R250 Brilliant blue

 

 

 

 

Destain

10% Acetic Acid

20% Methanol

 

For 1 liter:

200 ml Methanol

100 ml Glacial Acetic Acid

Fill to 1L with water

 

 

 

 

PHEM Buffer

5 mM HEPES

60 mM PIPES

10 mM EGTA

2 mM MgCl2

pH 7.0 with KOH

 

Prepare as a 2X solution and store at 4°C:

18.14 g PIPES

6.5 g HEPES

3.8 g EGTA

0.99 g MgSO4

 

 

 

 

 

Mounting Media for Immunofluorescence

20 mM Tris pH 8

90% Glycerol

0.5% N-phenyleindiamine

 

Aliquot and store protected from light at -80°C

 

 

 

 

Phosphate Buffered Saline (PBS)

137 mM NaCl

2.7 mM KCl

10.5 mM Na2HPO4

1.76 mM KH2PO4

pH of 1x = 7.4

 

To prepare 1 liter of 10x:

80 g NaCl

2 g KCl

11.5 g Na2HPO4 (MW 141.96)

2.4 g KH2PO4   (MW 136.09)

 

 

 

 

LB (Luria Broth)

10 g Tryptone

5 g Yeast extract

10 g NaCl

 

 

 

 

SOB Bacterial Media

(see http://en.wikipedia.org/wiki/Super_Optimal_Broth)

2% Bact-tryptone

0.5% Yeast extract

10 mM NaCl

2.5 mM KCl

 

To prepare 1 liter:

Measure ~900 ml of distilled H2O

Add 20 g Bacto Tryptone

Add 5 g Bacto Yeast Extract

Add 2 ml of 5M NaCl

Add 2.5 ml of 1M KCl

Adjust to 1 L with distilled H2O

 

Sterilize by autoclaving

 

 

 

 

SOC Bacterial Media

(see http://en.wikipedia.org/wiki/Super_Optimal_Broth)

 

2% Bact-tryptone

0.5% Yeast extract

10 mM NaCl

2.5 mM KCl

10 mM MgCl2 (or 20mM MgSO4)

20 mM Glucose

 

NOTE:

1- SOC is SOB + Glucose + MgCl2

2- Do not autoclave SOC!

 

To prepare 50 ml:

48.5 ml SOB

1ml 1 M Glucose

500ul 1 M MgCl2

 

 

 

 

Ponceau S

0.1% (w/v) Ponceau S

5% (v/v) acetic Acid

 

To prepare 200ml:

10 ml Glacial Acetic Acid

190 ml water

0.2 g Ponceau S

 

 

 

 

Tris Buffered Saline (TBS)

137 mM NaCl

2.7 mM KCl

25mM Tris

pH of 1x = 7.4

 

To prepare 1 liter of 10x:

80 g NaCl

2g KCl

30g Tris base   (MW  121)

 

 

 

 

 

TAE (Tris-acetate-EDTA) DNA gel running buffer

40 mM Tris

20 mM Acetic Acid

1mM EDTA

 

To prepare 1 L of 50x stock:

242 g Tris Base

18.6 g EDTA (or 100ml of 0.5M EDTA)

57.1 ml Glacial Acetic Acid

 

 

 

 

RIPA Lysis Buffer

50 mM Tris pH 7.5

150 mM NaCl

0.1% SDS

0.5% Na Deoxycholate

1% Triton-X 100

1 mM EDTA

 

To make 500 ml:

25 ml of 1M Tris, pH 7.5

15 ml 5M NaCl

500 µl of 10% SDS

2.5 g Na Deoxycholate

25 ml 20% Triton X 100

1 ml 0.5M EDTA

 

 

 

 

1KB DNA Ladder (working stock)

1 KB DNA ladder (Invitrogen cat no 10787-018; 250ul @ conc 1ug/ul) diluted to 0.2ug/ul

10 mM Tris pH 7.5

1 mM EDTA

50 mM NaCl

2x Orange G loading buffer

 

Prepare 1.25 ml stock:

250 ul 10x Orange G loading Buffer

250 ul 1KB DNA ladder

12.5 ul 5M NaCl

737.5 ul TE (Tris-EDTA)

 

Aliquot into ~200ul aliquots. Store working solution at 4°C, remaining aliquots at -20°C

 

 

 

 

TE (Tris-EDTA)

10 mM Tris pH 8

1 mM EDTA

 

 

 

 

10x Orange G DNA Loading Buffer

15% glycerol

2 mg/ml Orange G

 

Prepare 50 ml stock:

100 mg Orange G

7.5 mL Glycerol

Water to 50 mL

 

 

 

 

Ampicillin

Prepare100 mg/ml stock in water (store @ -20°C)

 

 

 

 

Spectinomycin

Prepare100 mg/ml stock in water (store @ -20°C)

 

 

 

 

Chloramphenical

Prepare 25 mg/ml stock in ethanol (store @ -20°C)

 

 

 

AEBSF (PMSF alternative- more stable)

Prepare 200 mM stock water (store @ -20°C)

 

 

 

 

Leupeptin (protease inh)

Dissolve to 10 mg/ml in ethanol

 

 

 

 

Aprotonin (protease inh)

Dissolve to 10 mg/ml in water

 

 

 

Pepstatin A (protease inh)

Dissolve to 10 mg/ml in DMSO or ethanol

 

 

 

 

Sodium Orthovanadate

Prepare 200mM stock in water.

To prepare, mix sodium orthvanadate with water and adjust pH to 10. Put in capped tubes and sit in boiling water for 10 minutes (or until solution becomes colorless). Repeat 2 more times, or until pH does not changes after heating. This process activates sodium orthovanadate.

 

 

 

 

Beta-glycerophosphate

Prepare 200mM stock solution in water and store at 4°C.

 

 

 

 

Sodium Fluoride

Prepare 200 mM stock in water and store at room temperature.

 

 

 

 

Hypotonic Lysis Buffer

10 mM HEPES

1.5 mM MgCl2

10 mM KCl

0.5 mM DTT