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 1 L

or

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

 

Stock B: 0.1 M Na2CO3 (fw 106)

10.6 g 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 (Marcs 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