Paul.xu at 15:25, 10 July 2019

2019-07-10T15:25:26Z

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Transform a sequence-verified expression plasmid into the expression host using heat shock, and plate the transformed cells onto agar plates of non-inducing medium. Place the freshly transformed cells in a 37°C incubator.

After 24 to 36 hr, pick a single colony from the agar plate and inoculate into 3 ml of non-inducing medium prewarmed to 37°C in a sterile 16-ml snap-top growth tube. Loosely cap the tube, place it in a 37°C incubator, and shake it at 300 rpm.

After 7 hr, add the 3-ml culture to 100 ml of non-inducing medium prewarmed to 37°C in a 500-ml Erlenmeyer flask. Loosely cap the flask, place it in a 25°C incubator, and shake it at 250 rpm.

After 24 hr, transfer 25 ml of the 100 ml culture to each of four 2-liter polyethyleneterepthalate beverage bottles containing 475 ml of autoinduction medium prewarmed to 25°C. Place the four separate culture bottles in a 25°C incubator, and grow cells with shaking at 250 rpm.

After 24 hr, harvest the cells by centrifuging 20 min at 4000 × g, 4°C in an Allegra 6R centrifuge with a GH3.8 rotor or equivalent, resuspend the cells in 30 ml of cell suspension buffer, and transfer the cell suspension to a 50-ml centrifuge tube.

Bring the volume of the cell suspension to 50 ml with cell suspension buffer and pellet the cells by centrifuging using the same conditions stated above. Store the washed and pelleted cells indefinitely at −80°C.

Perform analyses for expression by enzymatic assay, denaturing electrophoresis, or other methods depending on the nature of the protein and interests of the investigator.

== ZYP-5052 medium for auto-induction ==

This medium should be used when stronger buffering is needed. The amount of kanamycin (when appropriate) used should be at least 200µg/ml. Better even would be 400 µg/ml.

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| 
| 
| 
| Final
|-
| 928 ml 
| ZY 
|
tryptone

yeast extract

|
1% 

0.5%

|-
| 50 ml 
|  20 x P 
|
Na2HPO4 

KH2PO4 

(NH4)2SO4 

|
50 mM 

50 mM

25 mM

|-
| 20 ml 
|  50 x 5052 
|
 glycerol 

glucose 

alpha-lactose 

|
0.5% 

0.05% 

0.2%

|-
| 2 ml 
|  1 M MgSO4 
| MgSO4 
| 2 mM 
|-
| 0.2 ml 
| 1000 x trace elements 
| trace elements 
| 0.2X
|}

=== ===

== ZYM-505 medium for plasmid preparation ==

{| border="1" cellspacing="1" cellpadding="1" style="width: 500px;"
|-
| 
| 
| 
| Final
|-
| 968 ml 
| ZY 
|
tryptone

yeast extract

|
1% 

0.5%

|-
| 20 ml 
|  50 x M 
|
Na2HPO4 

KH2PO4 

(NH4)2SO4 

Na2SO4

|
25 mM 

25 mM

50 mM

5 mM

|-
| 10 ml 
|  50 x 505 
|
glycerol 

glucose 

|
0.5% 

0.05% 

|-
| 2 ml 
|  1 M MgSO4 
| MgSO4 
| 2 mM 
|-
| 0.2 ml 
| 1000 x trace elements 
| trace elements 
| 0.2X 
|}

=== '''ZY''' ===

1% tryptone: 10 g/L

0.5% yeast extract:  5 g/L

=== '''50 x M''' ===

1.25 M Na2HPO4-7H2O 335 g/L

1.25 M KH2PO4 170 g/L

2.5 M NH4Cl 134 g/L

0.25 M Na2SO4 35.5 g/L

Add in sequence to 700 ml water and stir until all salts have been dissolved.Occasionally crystals appear but they can be re-dissolved in the microwave. ThepH of a 20-fold dilution in water should be ~6.75.

=== '''100 x 5055''' ===

glycerol 500 g/L

glucose 50 g/L

=== '''20 x P''' ===

1.0 M Na2HPO4-7H2O 268 g/L

1.0 M KH2PO4 136 g/L

0.5 M (NH4)2SO4 66 g/L

Add in sequence to 770 ml water and stir until all salts have been dissolved. The pH of a 20-fold dilution in water should be ~6.75.

=== '''50 x 5052''' ===

25% glycerol 250 g/L

2.5% glucose 25 g/L

10% alpha-lactose 100 g/L  α-lactose

Add in sequence to 730 ml water and stir until all components have been dissolved. Lactose is slow to dissolve. It may take two hours or more at room temperature. The process can be sped up by heating in microwave.

=== '''1000x trace elements''' ===

50 mM FeCl3

20 mM CaCl2

10 mM MnCl2

10 mM ZnSO4

2 mM CoCl2

2 mM CuCl2

2 mM NiCl2

2 mM Na2MoO4

2 mM Na2SeO3

2 mM H3BO3

Preparation of 100 ml 1000x trace elements stock solution (in ~60 mM HCl).

Prepare stock solutions of the salts (except FeCl3) as mentioned in the table below.

Dissolve 0.1 M FeCl3 in 50 ml of 100x diluted concentrated HCl.

Add to 36 ml sterile water: 50 ml 0.1 M FeCl3-6H2O 2.70 g/100 ml

2 ml 1.0 M CaCl2-2H2O 15.8 g/100 ml

1 ml 1.0 M MnCl2-4H2O 19.8 g/100 ml

1 ml 1.0 M ZnSO4-7H2O 28.8 g/100 ml

1 ml 0.2 M CoCl2-6H2O 4.76 g/100 ml

2 ml 0.1 M CuCl2-2H2O 1.70 g/100 ml

1 ml 0.2 M NiCl2-6H2O 4.76 g/100 ml

2 ml 0.1 M Na2MoO4-2H2O 2.42 g/100 ml

2 ml 0.1 M Na2SeO3 1.73 g/100 ml

2 ml 0.1 M H3BO3 0.62 g/100 ml

{| border="1" cellspacing="1" cellpadding="1" style="width: 500px;"
|-
| 
| 
| 
| Final
|-
| 928 ml 
| ZY 
|
tryptone

yeast extract

|
1% 

0.5%

|-
| 50 ml 
|  20 x P 
|
Na2HPO4 

KH2PO4 

(NH4)2SO4 

|
50 mM 

50 mM

25 mM

|-
| 20 ml 
|  50 x 5052 
|
 glycerol 

glucose 

alpha-lactose 

|
0.5% 

0.05% 

0.2%

|-
| 2 ml 
|  1 M MgSO4 
| MgSO4 
| 2 mM 
|-
| 0.2 ml 
| 1000 x trace elements 
| trace elements 
| 0.2X 
|}
[[Category:Protocols]]

Protein Expression By Autoinduction - Revision history

Paul.xu at 15:25, 10 July 2019