Promega Protein Expression

S30 T7 High-Yield Protein Expression System Protocol

1, Remove reagents from freezer and thaw on ice (DNA tempplate, S30 Premix Plus, S30 Extract Circular, Nuclease-Free water)

aliquot 18μl T7 S30 Extract, to 500μl tube, store at - 80 oC freezer. 

2, In a 1.5 mL centrifuge tube, standard reaction:

DNA template Circular, 1μg

S30 Premix Plus,  20μl

T7 S30 Extract, 18 μl 

Add Nuclease-Free water to final volume of 50 μl in total

3, Pipette several times to mix

4, Centrifuge for 5 seconds

5, Put into 37 degree shaker overnight

6, Place in ice for 2 min. Add 50 ul cold Lysis buffer.

7, Transfer 50 ul of 10% Nickel magnetic beads to a tube,  put on a magnetic rack,  remove suspension buffer  using 200 ul pipet tip.

Add in vitro translation reaction solution, mix, incubate on ice, mix gently by swireling, mix gentely every 30 seconds, incubate for 5 min for efficient binding, put on a magnetic rack, transfer liquid to another tube using 200 ul pipet tip

8, Wash 3 X 200 ul  of Lysis Buffer, put on a magnetic rack, remove liquid using 200 ul pipet tip

9, Wash 1 X Washing Buffer, put on a magnetic rack, remove liquid using 200 ul pipet tip

10, Add 25 ul Buffer B, mix,  incubate on ice for 1 min

11, Put onto magnetic rack, transfer liquid to new tube.

12, repeat step 10, 11, combine liquid

13. Measure protein concentration with nanodrop, using Buffer B as blank , usually 2 mg/ml

14, Add equal volume of cold 80% glycerol, mix gentely, 

  optionally, aliquot 10 ug to clean tubes.

15, Print label and store protein in -80 oC freezer

Lysis Buffer:

0.5% NP-40, 10 mM Imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 8.0

Washing Buffer:  

1.5 M NaCl, 20 mM Tris-HCl, pH8.0

Buffer B:

10% glycerol, 500 mM Imidazole, 0.5 M NaCl, 1 mM DTT, 20 mM Tris-HCl, pH8.0