Kirschner Lab Buffers

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10x PBS 100 mL pH 9.2

8 g NaCl

0.2 g KCl

1.15 g Na2HPO4

0.2 g KH2PO4

pH to 9.2 with NaOH

50 mM Sodium Bicarbonate

0.042 g in 10 mL DI H2O

pH to 9.5 with NaOH

Rhodamine-B (5 mg / mL 5x pH 9.7-10)

0.025 g in 5 mL 50 mM Sodium Bicarbonate (pH 9.7 – 10)

Store at 4C wrapped in foil

50 mM Sodium Bicarb Buffered Saline (BBS) 1 L

Stock A: 0.1 M NaHCO3 (fw 84.01)

8.4 g à 1 L

or

150 mL of 750 mM NaHCO3 à 1125 mL (pH 8.4)

Stock B: 0.1 M Na2CO3 (fw 106)

10.6 g à 1 L

BBS pH 8.5 (1 L)

500 mL Stock A

~2 mL Stock B (pH to 8.5)

8.5 g NaCl

DI H2O to 1 L

BBS pH 9.3 (1 L)

400 mL Stock A

~100 mL Stock B (pH to 9.3)

8.5 g NaCl

DI H2O to 1 L

2 M NH4Cl (10 mL)

1.06 g to 10 mL DI H2O

Filter

Prehybridization mix (40 mL)

1 % BSA 10 mL 4 % BSA

1 % SDS 2 mL 20 % SDS

0.5 M NaPO4 (pH 7.9) 20 mL 1 M NaPO4 (ph 7.9)

1 mM EDTA 400 uL 100 mM EDTA (pH 8)

100 ug / mL carrier DNA 400 uL 10 mg / mL salmon sperm DNA

7.2 mL DI H2O

Store frozen (-20C)

Hybridization mix

1 vol kinase reaction

100 vol prehybridization mix

Acrylamide / Bis (30 % / 0.8 %) (200 mL)

60 g electrophoresis grade acrylamide

1.65 g bis (electrophoresis grade)

DI H2O to 200 mL

Stir

Store at 4C wrapped in foil

Use with 10 % (wt/vol) persulfate made fresh

0.1 g persulfate in 1 mL DI H2O

Laemmli Sample Buffer (10 mL 5X)

2.5 mL 1.25 M Tris (pH 6.8)

5 mL 100 % glycerol

1 g SDS (electrophoresis grade)

0.8 g DTT (electrophoresis grade)

1 mL Bromophenol Blue (1 % solution)

DI H2O to 10 mL (5X)

Laemmli Gel Buffers

Buffer I (8X 3 M pH 8.8 200 mL)

18 g Tris.HCl

58.8 g Tris Base

DI H2O to 200 mL

pH to 8.8

Buffer II (8X 1M pH 6.8 100 mL)

14.4 g Tris.HCl

1.3 g Tris Base

DI H2O to 100 mL

pH to 6.8

Laemmli Running Buffer (5X 1 L)

15.14 g Tris (mw 121.1)

72.1 g Glycine (mw 75.1)

5 g Lauryl Sulfate

DI H2O to 1 L

Store at 4C

HO Buffer without BME

250 mM NaCl

10 mM Tris (pH 7.4)

Fluorescein Working Stock (10 mg / mL 10X pH 9.7 - 10)

10 mg / mL fluorescein in

50 mM Sodium bicarbonate pH 9.7 - 10

Alcoholic KOH for plate washing

In 500 mL beaker, cover bottom with KOH pellets

~150 mL DI H2O to dissolve KOH

~250 mL ETOH (95 %)

Dialysis Buffer (1 L)

150 mM NaCl 8.8 g

15 mM MgCl 15 mL 1M MgCl (or 3.04 g)

10 mM Tris (pH 7.4) 10 mL 1 M Tris (pH 7.4)

DI H2O to 1 L

Stabilization Buffer for Microtubules

Stock A: 1 M PIPES (pH ~ 7.3)

33.5 g PIPES in 100 mL DI H2O

Stock B: 0.1 M EGTA (pH ~ 6.9)

1.9 g EGTA

15 mL DI H2O

pH with 1 M KOH

add DI H2O to 50 mL

Stabilization Buffer (10 mL)

1 mL Stock A (0.1 M PIPES)

0.1 mL Stock B (1 mM EGTA)

0.4 g PEG 8000 (4 % Polyethylene Glycol)

13 mg GTP (Na salt) (2.5 mM GTP)

+/- 5 uL / mL Triton X-100 (0.5 % Triton X-100)

PM2G Buffer (pH 6.9 1 L)

0.76 g EGTA (2 mM)

0.246 g MgSO4.7H2O (1 mM)

33.35 g PIPES (100 mM)

400 mL DI H2O

184.2 g Glycerol (2 M)

pH to 6.9 with 10 N NaOH

add DI H2O to 1 L

+ 0.1 % NP40 = 20 uL / mL (5 % NP40 stock)

+ PMSF = 1 uL / mL 0.2 M PMSF stock

PM Buffer (pH 6.9 100 mL)

3.34 g PIPES (100 mM)

0.025 g MgSO4.7H2O (1 mM)

1.47 g CaCl2 (1 mM)

50 mL DI H20

pH to 6.9 with 10 N NaOH

add DI H2O to 100 mL

GSD Buffer (6 mL 4X)

2 mL Glycerol

4 mL 10 % SDS

0.3 g DTT

Some Bromophenol Blue

Add DI H2O to 6 mL

TNT Buffer (6 L)

75 mL 2M Tris (pH 7.5)

59 g NaCl

30 mL Tween 20 (0.5 %)

Add DI H2O to 6 L

2 M Tris (pH 7.5 500 mL)

121 g Tris Base

300 mL DI H2O

pH with (lots) of HCl

Add DI H2O to 500 mL

PMSF (0.2 M 1000X 1 mL)

34.8 mg / mL in 95 % EtOH

Phenyl methyl sulfonyl fluoride (mw 174.2)

1 M Sodium Phosphate for blotting

1 L 1 M Di-basic Na2H(PO4)

500 mL 1 M Mono-basic NaH2(PO4)

Dilute to 20 mM for transfer

CsCl (density gradient)

74.2 g / 100 mL CsCl (1.546 g / mL)

37.1 g / 50 mL CsCl (1.546 g / mL)

61 g / 100 mL CsCl (1.45 g / mL)

27.7 g / 100 mL CsCl (1.2 g / mL)

Use DI H2O or Dialysis Buffer

Elisa III Buffer

15 mL 1 M NaCl (150 mM NaCl)

1 mL 100 mM EDTA (1 mM EDTA)

5 mL 1 M Tris (pH 7.4) (50 mM Tris.HCl)

50 uL Tween 20 (0.05 % Tween 20)

Adjust pH to 7.4

Add DI H2O to 100 mL

Add BSA to 0.1 %

1 M MOPS (pH 7.5 400 mL)

83.72 g MOPS

250 mL DI H2O

pH to 7.5 with 10 N NaOH

Add DI H2O to 400 mL

Fix for Antigen Staining

(PBS, 10 % sucrose, 3.7 % formaldehyde)

5 mL 10 X PBS

5 g sucrose

5 mL 37 % formaldehyde

Add DI H2O to 50 mL

DAB for Westerns (30 mL)

3 mL 10 % imidazol

30 mg DAB

30 uL 30 % H2O2 (Hydrogen peroxide)

Add DI H2O to 30 mL

1 M Tris (pH 7.4 500 mL)

60.5 g Tris Base

350 mL DI H2O

pH with HCl to pH 7.4 (~50 mL)

Add DI H2O to 500 mL

Completed Lysis Buffer for PI3K (100 mL)

4.57 mL 3 M NaCl (137 mM NaCl)

2 mL 1 M Tris (pH 7.5) (20 mM Tris)

0.1 mL 1 M MgCl2 (1 mM MgCl2)

0.1 mL 1 M CaCl2 (1 mM CaCl2)

10 mL 100 % glycerol (10 % glycerol)

1 mL NP-40 (1 % NP-40)

Add DI H2O to 100 mL

Store at 4C.

Just before use, add protease inhibitors.

Poly-lysine – Laminin Coating for cover slips

10 mL poly-lysine

79 uL laminin

3.78 mL Sterile DI H2O

100 – 150 uL per cover slip (12 mm round)

1 hr at RT

Remove poly-lysine/laminin

Wash 2 X with Sterile DI H2O

Leave wet until use

1 X Semi-Dry Western Transfer Buffer

2.93 g Glycine (39 mM Glycine)

5.81 g Tris-Base (48 mM Tris)

0.375 g SDS

100 mL DI H2O

200 mL Methanol

Add DI H2O to 1 L

Mono Q buffers

QA (2 L: 20 mM Tris pH 7.7, 100 mM KCl, 1 mM MgCl2, 1 mM DTT)

40 mL 1 M Tris (pH 7.7)

14.9 g KCl

0.406 g MgCl2

2 mL 1 M DTT (1.54g DTT in 10 mL DI H2O = 1 M DTT)

Add DI H2O to 2 L

QA (alt)

1 X XB Salts (20 X Stock)

20 mM Tris-HCl (pH 7.7)

Degas before using on FPLC

QB (1 L QA + 0.9 M KCl)

1 L QA

67.1 g KCl

QB (alt)

1 L QA (alt)

141.6 g KCl (1.9 M KCl)

Degas before using on FPLC

TAE Buffer (50 X stock 1 L)

242 g Tris Base

57.1 mL Glacial Acetic Acid

100 mL 0.5 M EDTA (pH 8.0)

Add DI H2O to 1 L

PBS (1 X 100 mL)

0.8 g NaCl

0.2 g KCl

0.115 g Na2HPO4

0.02 g KH2PO4 pH ~7.1-7.2

Filter Sterilize

Carbenicilin (100 X)

10 mg / mL Carbenicilin

Filter sterilize, then freeze/store 1 mL aliquots

20 X SSC

(3 M NaCl, 0.3 M Na3Citrate)

175.3 g NaCl

88.2 g Na3Citrate

800 mL DIH2O

Adjust pH to 7.0 with 1 M HCl

Add DI H2O to 1 L

Kanamycin (1000 X)

30 mg / mL Kanamycin

Filter sterilize, then freeze/store 1 mL aliquots

1000 X stock for pET vectors

600 X for BactoBAC cells

IPTG (1 M Stock)

2.4 g IPTG

10 mL DI H2O

Filter sterilize, then freeze/store 1 mL aliquots

0.4 - 1.0 mM final working concentration (Stock is 1000 – 2500 X)

Lysis Buffer for sf9/baculo cells

20 mL 1 X TBS

20 uL Triton X-100 (0.1 % Triton X-100)

2 X Sample Buffer (10 mL)

1 mL 1 M Tris (pH 6.8)

2 mL 20 % SDS

2 mL 100 % Glycerol

2 mL 1 % Bromophenol Blue

3 mL DI H2O

Add 0.2 mL 1 M DTT prior to use

Buffer for S300 Column (1 L for CDC16)

20 mL 1 M Tris (pH 7.7)

7.45 g KCl

1 mL 1 M DTT

Optional: add 1 mL of 1 M MgCl2

Add DI H2O to 1 L

Stacking Gel Solution

8 mL Acrylamide:bis 37.5:1

7.5 mL 1 M Tris (pH 6.8)

0.3 mL 20 % SDS

44.4 mL DI H2O

Makes 50 mL to be stored at 4C

For each 5 mL (one gel): add 50 uL 10 % APS (ammonium persulfate)

and 5 uL TEMED

Western Strip Buffer (100 mL)

6.25 mL 1 M Tris (pH 6.7)

10 mL 20 % SDS

0.7 mL BME (beta-Mercapto Ethanol)

Add DI H2O to 100 mL

Wash Blot 30 min at 50C

0.5 M Imidazole for bead elution (100 mL)

3.4 g Imidazole

1 mL 0.5 M NaHPO4 (pH 6)

3.3 mL 1 M KCl

Add DI H2O to 100 mL

0.5 M NaHPO4 (pH 6 30 mL)

20 mL 0.5 M NaH2PO4 (pH ~4.3)

10 mL 0.5 M Na2HPO4 (pH ~7.2)

0.5 M NaH2PO4 (100 mL mw 120)

6 g NaH2PO4

100 mL DI H2O

0.5 M Na2HPO4 (100 ml mw 142)

7.1 g Na2HPO4

100 mL DI H2O

1 M DTT (10 mL Stock fw 154.2)

1.542 g DTT

10 mL DI H2O

Aliquot and store at -20C

20 X PBS (500 mL)

80 g NaCl

20 g KCl

11.5 g Na2HPO4

2 g KH2PO4 (pH 7.1-7.2)

Add DI H2O to 500 mL and filter sterilize

4 % paraformaldehyde (100 mL)

4 g paraformaldehyde

100 mL hot (60 – 65C) DI H2O

100 mM KxFe(CN)6 (100 mL)

4.2 g K4Fe(CN)6

3.3 g K3Fe(CN)6

100 uL 1 M MgCl2

100 mL 1 X PBS

6M Gu-HCl, 0.1 M Na Phosphate, 0.01 M Tris-HCl (pH 8) (50 mL)

28.7 g GuHCl (fw 95.53)

10 mL 0.5 M NaHPO4 (pH 7.2)

0.5 mL 1 M Tris (pH 8.0)

Add DI H2O to 50 mL

Dialysis Buffer for APC urea Dialysis (1.5 L)

15 mL 1 M Tris (pH 7.4) (10 mM Tris, pH 7.4)

26.5 g NaCl (300 mM NaCl)

0.3 g MgCl2 (1 mM MgCl2)

1.5 mL 1 M DTT (1 mM DTT)

3 mL 0.5 M EDTA (1 mM EDTA)

0.22 g CaCl2 (1 mM CaCl2)

0.15 g BSA (0.1 mg/mL BSA)

180 g Urea (2 M Urea)

or

90 g Urea (1 M Urea)

XB Extraction Buffer (20 X 1 X)

20 X Salts (2 M KCl, 20 mM MgCl2, 2 mM CaCl2)

1 X Salts (100 mM KCl, 1 mM MgCl2, 0.1 mM CaCl2)

1 X (1 X Salts, 10 mM Potassium HEPES (pH 7.7), 50 mM Sucrose)

20 X XB Salts Buffer (500 mL)

74.55 g KCl

0.147 g CaCl2-2H2O

2 g MgCl2

Add DI H2O to 500 mL

PIN-1 Wash Buffer (20 mL)

1 mL 1 M Tris (pH 8.0)

2 mL 2 M NaCl

4 mL 0.5 M NaF

2 mL 100 % Glycerol

0.2 mL Triton X-100

0.4 mL 0.5 M EDTA (pH 8.0)

1 uM microcystin (okadaic acid)

1 X protease inhibitors

Cyclohexamide (100 X Stock)

10 mg / mL Cyclohexamide in DI H2O

Store frozen aliquots at -80C

Energy Mix (20 X 5 mL)

0.245 g Creatine Phosphate

1 mL 100 mM ATP

100 uL 1 M MgCl2

Add DI H2O to 5 mL

Store in aliquots at -20C

EB (1 X 400 mL)

6.912 g glycerophosphate (80 mM)

3.044 g EGTA (20 mM)

1.22 g MgCl2 (15 mM)

300 mL DI H2O

pH to 7.8 with KOH

Add DI H2O to 400 mL

Sperm Dilution Buffer (1 x 5 mL)

5 uL 1 M MgCl2 (1 mM MgCl2)

0.5 mL 1 M KCl (100 mM KCl)

375 uL 2 M Sucrose (150 mM Sucrose)

25 uL 1 M HEPES (pH 7.7) (5 mM HEPES)

4.1 mL DI H2O

Fix for Nuclei (make fresh)

0.3 vol 37 % formaldehyde

0.6 vol 80 % w/v glycerol

0.1 vol 10 X MMR

1 ug / mL Hoechst dye bis benzimide (10 mg / mL stock at -20C)

CSF-XB (100 mL)

5 mL 20 X XB Salts (1 X Salts)

1 mL 1 M HEPES (10 mM HEPES)

2.5 mL 2 M Sucrose (50 mM Sucrose)

1 mL 0.5 M EGTA (5 mM EGTA)

2 X – XN Buffer (100 mL)

10 mL 1 M HEPES (KOH, pH 7.0) (100 mM HEPES)

10 mL 2 M Sucrose (200 mM Sucrose)

7.5 mL 2 M NaCl (150 mM NaCl)

25.4 mg Spermidine-4HCl (1 mM Spermidine)

10.44 mg Spermine-4HCl (0.3 mM Spermine)

Add DI H2O to 100 mL

G-PEM Buffer for rho tubulin

80 mM PIPES (pH 6.8)

1 mM MgCl2

1 mM EGTA

1 mM GTP

10 % glycerol

0.5 M EGTA (100 mL pH 7.7)

19 g EGTA

70 mL DI H2O

pH to 7.7 with 10 N NaOH (~10-15 mL)

Add DI H2O to 100 mL

25 X MMR (Marc’s Modified Ringers 2 L pH 7.8)

292.2 g NaCl

7.45 g KCl

10.15 g MgCl2

14.7 g CaCl2

1.8 g EDTA

59.6 g HEPES

1.2 L DI H2O

pH to 7.8 with NaOH

Add DI H2O to 2 L

Nuclei Isolation Buffer A (100 mL)

(60 mM KCl, 15 mM NaCl, 0.15 mM Spermine, 0.5 mM Spermidine,

15 mM Tris (pH 7.4), 0.2 mM EDTA, 0.2 mM EGTA)

6 mL 1 M KCl

1.5 mL 1 M Tris (pH 7.4)

0.5 mL 3 M NaCl

40 uL 0.5 M EDTA

40 uL 0.5 M EGTA

150 uL 100 mM Spermine

250 uL 200 mM Spermidine

0.5 M Na pyrophosphate (dibasic) (20 mL pH 7.0)

2.21 g / 20 mL

pH to 7.0 with phosphoric acid

Stop Buffer (or Stabilization Buffer) SB (10 mL pH 7.0)

(50 mM NaF, 40 mM beta-glycerophosphate, 10 mM EDTA,

10 mM Na Pyrophosphate pH 7.0)

1 mL 0.5 M NaF

0.4 mL 1 M beta-glycerophosphate

0.2 mL 0.5 M EDTA

0.2 mL 0.5 M Na2HP2O7

2 X YT (per liter)

16 g bactotryptone

10 g bacto yeast

5 g NaCl

pH to 7.0 with 5 N NaOH

Wash Buffer for GST Purification (200 mL pH 8.2)

10 mL 1 M Tris (pH 8.2) (50 mM Tris)

2.96 g KCl (200 mM)

0.2 mL 1 M DTT (1 mM DTT)

Add protease inhibitors before use

Lysis Buffer for GST Purification

Wash Buffer for GST Purification + 0.3 mM PMSF

+ 200 ug / mL lysozyme

10x Taq/Pfu Buffer

(from Teresita/Ethan August 2003)

200 mM Tris HCL

20 mM MgSO₄

100 mM KCl

100 mM (NH4)₂ SO₄

1 mg/ml Nuclease-free BSA

FILTER
-------------------------------------

1% Triton X-100

AMINO ACIDS

(from Teresita/Ethan September 2001)

Amino Acid Mix (minus Methionine, Cysteine, Cystine)

• Assume 110g/ mole for each amino acid.
• Want to add enough to make 2mM stock

110g/ X = 1 M/ 0.002M (= 2 mM)

therefore X= 220 mg/ liter

• To make 50 ml
• Add 11 mg of each amino acid EXCEPT: METHIONINE, CYSTEINE, CYSTINE
• Bring to 40 ml with sterile water, pH to 7.0 with either HCL or NaOH add water to 50 ml in a sterile beaker/tube.

2 mM Methionine

11 mg Methionine

40 ml H₂0

pH to 7.0

add H₂0 to 50 ml

2 mM Cysteine/ Cystine

11 mg Cysteine

11 mg Cystine

40 ml H₂0

pH to 7.0

H₂0 to 50 ml

L-Amino Acids (SIGMA LAA-21) – 1 Kit

Translation Reaction Mix

(Teresita/Ethan May 2002)

Promega

Per Reaction (5 μl)

TnT Retic lysate 2.5 μl

Tnt Reaction buffer 0.2 μl

RNA pol. SP6 0.1 μl

AA (minus Met) 0.1 μl

S³⁵ Methionine 0.4 μl

Rnasin 0.1 μl

H₂0 1.6 μl

5.0μl

IVT

1) 5 μl TnT Mix + 1 μl DNA

2) Incubate 1 hour @ 30 ⁰C

Homemade (5X E/T Buffer)

Per Reaction (5 μl)

Retic lysate* 2.5 μl

5X Reaction buffer 1 μl

RNA pol. SP6 0.1 μl

AA (minus Met/ Cys) 0.1 μl

S³⁵ (Met/Cys) 0.4 μl

Rnasin 0.1 μl

H₂0 0.8 μl

5.0μl

*Promega Catalog # L4960

Enhanced Chemiluminescence (ECL)2

(Teresita/ Licio/ Ethan May 2002)

Stock Solution:

250 mM Luminol (in DMSO)

90 mM p-Coumaric acid in (DMSO)

30% Hydrogen peroxide (H₂0₂)

100 mM Tris-HCL (pH 8.5-9.0)

DMSO

Solution A (Final concentration):

0.0165% H₂O₂

100 mM Tris-HCL

Volumes for 20 (final) ml of ECL Buffers

Stock Solutions

100mM Tris-HCL

30 % H₂O₂

250 mM Luminol

90 mM p-Coumaric Acid

FLUKA 09253 5 g

SIGMA C-9008 10 g

SIGMA H-1009 5 ml

SIGMA D-2650 100 ml

Solution B (Final concentration):

2.5 mM Luminol

0.4 mM p-Coumaric acid

100 mM Tris-HCl

Solution A	Solution B
10 ml	10 ml
5.5 μl	--------
---------	100 μl
---------	45 μl

5X TnT Reaction Buffer

(Teresita/ Ethan May 2002)

Final Concentration:

50	mM	Potassium acetate
2	mM	Magnesium Chloride
0.05	mg/ml	Creatine kinase
1	mM	Creatine phosphate
1	mM	ATP
1	mM	CTP
1	mM	GTP
1	mM	UTP

Stock Solution:

1	M	Potassium Acetate	------
40	mM	Magnesium Chloride	------
1	Mg/ml	Creatine Kinase	ROCHE 127 566	100 mg
100	mM	Creatine phosphate	ROCHE 621 714	5 g
100	mM	ATP	SIGMA A-9062	100 mg
100	mM	CTP	SIGMA C-1631	100 mg
100	mM	GTP	SIGMA G-9002	100 mg
100	mM	UTP	SIGMA U-6875	100 mg