T7 Preparation

Buffers Needed:
Lysis Buffer:            100ml
50 mM Tris               2.24 ml 1M Tris Base; 2.76 ml 1M Tris HCl
 5 mM BME                35.06 uL  (** Add after autoclave)
300 mM NaCl              1.75 g
5% Glycerol              5 ml  100% glycerol
pH 8.0		

Wash Buffer:             250 ml
50 mM Tris               6.72 ml 1M Tris Base; 8.25 ml 1M Tris Hcl 
5 mM BME                 105.18 uL  (**Add after autoclave)
800 mM NaCl              11.69 g 
4 mM Imidazole           0.068 g 
5% Glycerol              12.5 ml  100%
pH 8.0

Elution Buffer:          100 ml
50 mM Tris               2.24 ml 1M Tris Base; 2.76 ml 1M Tris HCl
5 mM BME                 35.06 ul (**Add after autoclave)
800 mM NaCl              4.68 g
100 mM Imidazole         0.68 g
5% Glycerol              5 ml 100%
pH 8.0


Elution Buffer 2:
Same as above, 250 mM Imidazole.

Storage Buffer:          1L
20 mM Tris               9.98 ml 1M Tris Base; 10.02 ml 1M Tris HCl
100 mM NaCl              100 ml NaCl 1M
1 mM EDTA                2 ml    0.5 M EDTA
5% Glycerol              50 ml 100%
1 mM DTT                 1 ml 1M  (** Add after autoclave)
pH 8.0

Cell Growth:

Autoclave 6 x 100ml and 6 x 1L LB-ampicillin media (Amp stock: 1g amp/10ml add 1ml         
              amp stock/ 1L media )

Autoclave 6 x 500ml centrifuge bottles

Make and autoclave lysis buffer. 

Remove 6 x 5 ml media and put into culture tubes for overnight starter culture.  Inocuate with T7 BL21 cell stock.

Grow overnight

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Pour one 5 ml starter culture into 100 ml LB-amp media.

Grow until OD ~0.6 (4 hrs)

Put 100 ml cultures into 500ml centrifuge tube (split into ~300ml/tube)  
            Spin 5,000g 25 C 20 min.

Resuspend in fresh LB (take from the 1L flasks) and divide evenly among the 6 x 1L                                      
            flasks.  

Grow until and OD~0.6 (~3 hrs)

Induce over expression by adding 1ml 1M IPTG / 1L media.

Grow until max OD ~3-4 (3 hrs)

Spin 5,000g 25 C 20 min Pour off supernatant.

Resuspend each liter of cells in ~30 ml lysis buffer.  (Can freeze in -20 C freezer for later purification.)  DO NOT FREEZE IN 500 ml CENTRIFUGE BOTTLES- USE FALCON TUBES. 


Protein Purification:

Make and autoclave all buffers except lysis buffer.

Add Lysozyme to cells ~1 mg / g cells.  (each liter is about 4-5 g cells)  

Lyse cells in french press

Centrifuge 40 min 15,000 rpm.  Keep supernatant.

Equilibrate Co2+ resin (Talon Affinity Resin) in lysis buffer. 
         need 5 ml resin per liter cells.  Resin comes in 50% slurry therefore 10 ml slurry = 
        5 ml resin.  Allow resin to settle, (or spin) and pour off liquid.  Add 40 ml lysis  
         buffer,  shake, settle, pour off;  do this 3 x.

Check pH, should be 8 (that of lysis buffer).

Add lysate to resin and allow to bind for ~1.5 hr at 4 C.  Put on spinner in fridge or in ice
        bath on bench top shaker.

Let settle out, or spin and pour off supernatant and save.

Wash 6 x with 10 ml wash buffer. let settle and save all supernatant.

Check OD280; should be decreasing.  Very little T7 in super.

Elute T7 with 100 mM imidazole elution buffer: do 5 x 10 ml each time.  Let settle (or 
      spin and save supernatant)  This should contain most of the T7.    

Wash 1 x with 250 mM imidazole elution buffer 2 overnight to remove remaining tightly
        bound T7.

Dialyze into storage buffer. (note: exchange via centricons does not remove imidazole)