Basic Stock Solutions

(Dr. Jihun Lee, June 5 2007)

 

0.5M Na2HPO4 (dibasic) 

§     Vendor: Fisher, Cat# S374-1, FW=141.96

§     Dissolve 70.98gm Na2HPO4 in 800ml of ddH2O and make the volume to 1L and filter.

 

1.0M NaH2PO4. H2O (monobasic) 

§     Vendor: Fisher, Cat# S9638-1KG, FW=137.99

§     Dissolve 137.99gm NaH2PO4.H2O in 800ml of ddH2O and make the volume to 1L and filter.

 

0.5M EDTA stock

§     EDTA disodium salt

§     Vendor: Fisher, Cat# BP120-500, FW=372.24

§     Dissolve 93.06gm of EDTA in ddH2O and make the volume to 500ml.

 

0.1M EDTA stock

§     EDTA disodium salt

§     Vendor: Fisher, Cat# BP120-500, FW=372.24

§     Dissolve 37.22gm of EDTA in ddH2O and make the volume to 1L.

 

1.0M IPTG stock**

§     Vendor: Anatrace, Cat# I1003, FW=238.31

§     Dissolve 7.15gm of IPTG in 20ml of ddH2O and vortex it for some time. Make the final volume to 30ml and mix again. Aliquot in 1ml (x 30) in autoclaved epp. tubes.

** may take some time to dissolve (not very soluble in water)

 

Amp. 150  stock.

§     Vendor: Sigma, A9518-25G, FW=371.39

§     Dissolve 3.3 gm of Amp. in 22ml of water and syringe filter it. Aliquot in 1ml (x20) and 0.1ml (x20) in autoclaved epp. tubes .

§     Always use sodium salt of Amp.

 

20% D-Glucose

§     Same as Dextrose

§     Vendor: Fisher, Cat# D16-1, FW=180.16

§     Dissolve 80gm Glucose in 300ml of ddH2O and make the final volume to 400ml and filter. Use autoclaved bottle for storage.

 

0.1M NiSO4 

§     NiSO4 hexahydrate

§     Vendor: Sigma, Cat# N-4882, FW=262.8

§     Dissolve 26.28gm NiSO4 in 800ml of ddH2O and make the final volume to 1L and filter.

 

2%SDS

§     Vendor: Fisher, Cat# BP466-500, FW=288.38

§     Dissolve 20.0gm SDS in 800ml of ddH2O and make the final volume to 1L.

 

1.0M NaOH 

§     Vendor: Fisher, Cat# S318-500, FW=40.00

§     Dissolve 10gm NaOH in 200ml of ddH2O and make the final volume to 250ml.

 

10.0M NaOH 

§     Vendor: Fisher, Cat# S318-500, FW=40.00

§     Dissolve 100gm NaOH in 200ml of ddH2O and make the final volume to 250ml.

 

1.0M Sodium Acetate stock 

§     Sodium Acetate Trihydrate

§     Vendor: Fisher, Cat# S209-500, FW=136.08

§     Dissolve 136.08gm NaOAc in 800ml of ddH2O and make the final volume to 1L.

 

 

For E. Coli Expression: Media and plates

 

M9 media composition for IL**

 

Na2HPO4 (Diabasic)   :   12.0gm

KH2PO4                      :   3.0gm

NaCl                            :   0.5gm

NH4Cl                         :   1gm

ddH2O                        :   987ml

 

** must be autoclaved

 

  • Na2HPO4 (Diabasic) : Fisher, Cat# S374-1, FW=141.96
  • KH2PO4                    : Fisher, Cat# P285-3, FW=136.09
  • NaCl                          : Fisher, Cat# BP358-212, FW=58.44
  • NH4Cl                     : Fisher, Cat# A661-500, FW=53.49

 

1X YT plate Amp. 150

 

Trypton                                   : 6.4gm

Yeast extract                           : 4.0gm

NaCl                                        : 4.0gm

Bacto agar                               : 12gm

Amp. Stock (150mg/ml)         : 533ml

 

Mix every thing except Amp. And make the final volume to 800ml by ddH2O and autoclave it. Cool it to 50oC in the water bath and add Amp. and pour plates (~20 plates).

 

  • Trypton: BD, Cat# 211705
  • Yeast extract: Difco Laboratories, Cat# 0127-17-9
  • NaCl: Fisher, Cat# BP358-212, FW=58.44
  • Bacto agar: BD, Cat# 214010

 

Ni-NTA purification of hisFGF-1

Ni-NTA buffer

Buffer

Composition

Amount for 1L

pH

5mM imidazole

50mM NaPi

500mM NaCl

5mM imidazole

 

Dibasic (0.5M): 80.95ml and Monobasic (1M): 9.5ml

29.22gm

0.3404gm

 

7.5

50mM imidazole

50mM NaPi

500mM NaCl

50mM imidazole

 

Dibasic (0.5M): 80.95ml and Monobasic (1M): 9.5ml

29.22gm

3.404gm

 

7.5

250mM imidazole

50mM NaPi

500mM NaCl

250mM imidazole

 

Dibasic (0.5M): 80.95ml and Monobasic (1M): 9.5ml

29.22gm

17.02gm

 

7.5

* Adjust pH with either 10M NaOH or 1M NaOH

 

  • NaCl: Fisher, Cat# BP358-212, FW=58.44
  • Imidazole: Sigma, Cat# I2399-500G, FW=68.08

 

Ni-NTA regeneration buffer**

Buffer

Composition

Amount for 1L

Ni- regeneration buffer

 

6.0M GuHCl

0.2M acetic acid

 

573.18gm

11.5ml

 

** Dissolve all components the final volume 1L. Filter after preparation.

 

  • GuHCl: Heico Chemicals Inc. FW=95.53 (Note: Heico GuHCl may no longer be available.  Substitute with best quality alternative.)
  • HOAc: EMD, Cat# UN2789

 

Heparin column purification of hisFGF-1

Heparin Buffers**

Buffer

Composition

Amount for 1L

pH

Heparin Buffer

50mM NaPi

500mM NaCl

10mM (NH4)2SO4

2mM DTT

Dibasic (0.5M): 80.95ml and Monobasic (1M): 9.5ml

29.22gm

1.3214gm

0.3084gm

 

7.5

Heparin Elution Buffer

50mM NaPi

2M NaCl

10mM (NH4)2SO4

2mM DTT

Dibasic (0.5M): 80.95ml and Monobasic (1M): 9.5ml

116.88gm

1.3214gm

0.3084gm

 

7.5

** Occasionally we may ask heparin buffers without DTT

 

  • NaCl: Fisher, Cat# BP358-212, FW=58.44
  • (NH4)2SO4: Fisher, Cat# BP212-212, FW=132.14
  • DTT (Cleland’s Reagent): CALBIOCHEM, Cat# 233155, FW=154.2

 

Heparin regeneration buffer**

 

Buffer

Composition

Amount for 1L

pH

Heparin regeneration buffer

 

5.5M GuHCl

0.1M Sodium acetate

0.5M NaCl

 

525.415gm

100ml of 1M sodium acetate stock solution

29.22gm

 

 

5.3

** Dissolve all components set the pH and finally make the final volume 1L. Filter after preparation.

 

  • GuHCl: Heico Chemicals Inc. FW=95.53 (Note: Heico GuHCl may no longer be available.  Substitute with best quality alternative.)
  • NaCl: Fisher, Cat# BP358-212, FW=58.44

 

Buffers for Biophysical Study of hisFGF-1

 

ADA Buffer**

 

Buffer

Composition

Amount for 1L

pH

ADA Buffer

20mM ADA

100mM NaCl

2mM DTT

 

3.804gm

5.844gm

0.3084gm

 

 

6.6

** Occasionally we may ask ADA buffer without DTT

** Occasionally we may ask ADA buffer with 10mM (NH4)2SO4 (1.3214gm for 1L)

** Occasionally we may ask ADA buffer with 10mM (NH4)2SO4 (1.3214gm for 1L) without DTT

 

* ADA is not dissolved very well with initial pH ~2. It will be dissolved eventually by adding 10M NaOH to adjust pH 6.6.

 

  • ADA: Sigma, Cat# A8074-100G, FW=190.16
  • NaCl: Fisher, Cat# BP358-212, FW=58.44
  • DTT (Cleland’s Reagent): CALBIOCHEM, Cat# 233155, FW=154.2

 

Crystal buffer (X-tal buffer) **

Buffer

Composition

Amount for 1L

pH

X-tal buffer

50mM NaPi

100mM NaCl

10mM (NH4)2SO4

2mM DTT

0.5mM EDTA

Dibasic (0.5M): 80.95ml and Monobasic (1M): 9.5ml

5.844gm

1.3214gm

0.3084gm

1ml of 0.5M stock

 

7.5

* Adjust pH with either 10M NaOH or 1M NaOH

** Occasionally we may ask X-tal buffers without DTT

** Occasionally we may ask X-tal buffers without EDTA

** Occasionally we may ask X-tal buffers without DTT and EDTA

 

  • NaCl: Fisher, Cat# BP358-212, FW=58.44
  • (NH4)2SO4: Fisher, Cat# BP212-212, FW=132.14
  • DTT (Cleland’s Reagent): CALBIOCHEM, Cat# 233155, FW=154.2

© 2007 Blaber Lab