Editing Generic Protein Expression Screening Protocol (section)

== pCold Screening ==

1. Transform proper competent cells such as Nico21(DE3)/pLysN, Rosetta BL21(DE3)/pLysS, BL21 Star (DE3)/pRare2pLysS with plasmid.

2. Grow on an agar plate with proper selection antibiotics, such as K/Chl

3. In the morning, pick 3 colonies,&nbsp; grow them in '''5 ml or 15 ml'''&nbsp;LB/antibiotics (optional 0.5% Glucose, Glucose can suppress leaky/basal expression) in autoclaved glass tubes, at 37 oC for 4~6 hours (OD600=0.8~1.5).

4. Cool down to 25 oC using running water.

5. Add 100 uM or 1 M IPTG to final concentration of 0.1 to 0.2 mM. Induce at room termperature o/n. Make sure there is one uninduced tube for control.

6. Spin down cells

7. Add 400 ul of Lysis '''Buffer S''' to induced and uninduced cell pellets. Votex to suspend cells. Transfer the suspension to microcentrifuge tube by pouring.

'''Lysis Buffer S''': 10 mM Imidozale, 20 mM Tris-HCl, pH 8.0 &nbsp;or &nbsp;[[Phosphate_Buffer_Chart|50 mM Na-Phosphate, pH 7.4]], 300&nbsp; mM NaCl, 1 mM beta-ME, 0.1% Triton X-100, 1<strike>0% glycerol</strike>.

8. Freeze-thaw cells 1 to 3 cycles. If lysis does not occure, add 2~4 ul 100X lysozyme, incubate at 37 oC with shaking for 15~30 min until the cells are lysed.

9. If the lysate is too sticky (too much DNA), add 0.25 ul Benzonase. Incubate at 37 oC for another 15 min. Or use needle to break sticky DNA&nbsp;

10. Spin down pellet at 13000 rpm for 2 min. Collect supernatant to a new tube. Do not discard the tube with pellet.

<u>Steps 11 and 12 are optional:</u>

11. Add 200 ul Lysis '''Buffer P''' to pellet

'''Lysis Buffer P''': 10 mM Imidozale, 20 mM Tris-HCl pH 8.0, 600&nbsp;NaCl, &nbsp; [[Phosphate_Buffer_Chart|50 mM Na-Phosphate, pH 7.4,]] 300 mM NaCl, 1 mM beta-ME, 0.5% SDS or Triton X-100, '''8 M Urea'''. Mix by votex gently.

12. Spin down pellet at 13000 rpm for 2 min. Collect supernatant to a new tube.

13. Add 50 ul regenerated Ni beads (50% suspension) to both lysates.

14. Incubate with shaking or rotating for 15~ 30 min at RT.

15. Spin down beads gently (1000 rpm, 1 min). Transfer supernatant by pouring the liquid into the original tube at step 10.

16. Wash beads 3 times with 200 ul of &nbsp;'''Washing Buffer''': <u>35 mM Imidozale</u>, <strike>20 mM Tris-HCl pH 8.0, 600 mM NaCl</strike>,&nbsp;[[Phosphate_Buffer_Chart|50 mM Na-Phosphate, pH 7.4]], 300 mM NaCl, <u>4M Urea</u>, 1 mM beta-ME, 0.1% SDS, <strike>Triton X-100</strike>, <strike>10% glycerol</strike>. Spin down beads gently at 1000 rpm for 1 min, carefully remove washing solutions (do not pipette out beads).

17. Wash beads 1 time with 500 ul of &nbsp;'''Washing Buffer II''': 35 mM Imidozale,&nbsp;&nbsp; 50 mM Na-Phosphate, pH 7.4, 300 mM NaCl. Spin down beads gently at 1000 rpm for 1 min, carefully remove washing solutions (do not pipette out beads).

18. Add 20 ~ 30 ul '''Elution Buffer:''' <u>300 mM Imidozale</u>, 20 mM Tris-HCl, pH 8.0. Incubate at RT for at least 1 min.

19. Add 5 ul 4X or 6X SDS-Protein SDS-PAGE Loading Buffer to a row of PCR tubes (reuse, do not throw away).&nbsp; Transfer 15 ul eluate to individual tube.

20. Boil protein at 95 oC for 2 min. (Do not boil Ni beads, combine these used beads and store in a bottle with 20% ethanol)

21. Run SDS-PAGE.

22. Stain with SimplyBlue or other protein dyes.

23. Destain with water

24. Scan gel and upload gel image to ELN. Document all experimental conditions.

25. Make a glycerol stock of expression clones.