Editing Generic Protein Expression Screening Protocol (section)

== Transformation and Small Scale Expression Screening ==

1. If a&nbsp;clone in a vector&nbsp;has&nbsp;already been transformed into expression host such as BL21, Nico21, find the glycerol stock from -80 oC freezer, strick bacteria on LB/antibiotics plate (e.g., LB/KC). Grow o/n at -37 oC. Pick a few colonies and grow in LB/antibiotics liquid medium for a few hours, when OD600 &nbsp;is ~0.8~1, add 1 M IPTG to final concentration of &nbsp;0.2~0.5 mM. induce 4 hours at 37 oC or 25 ~30 oC o/m.



2. If there is no expression bacteria&nbsp;for the &nbsp;clone, grow up bacterial stock (such as DH5alpha, NEB Turbo) in 5 ml liquid medium with antibiotics overnight at 37 oC. Make miniprep, Transform expression host such as Nico21pLysN using&nbsp;'''0.5 ul '''of plasmid DNA. Grow on LB/antibiotics plate (such as LB/KC) overnight at 37 oC. Pick a few colonies and grow in 15 ml LB/antibiotics liquid for a few hours,&nbsp;, when OD600 &nbsp;is ~0.8~1, add 1 M IPTG to final concentration of &nbsp;0.2~0.5 mM. induce 4 hours at 37 oC or 25 ~30 oC o/m.



3. Transfer cell culture to &nbsp; 15 ml conical tube. Spin down cells at 6000 rpm for 5 min. Add 1ml lysis buffer. transfer to 1.5 ml microcentrifuge tube. Put into dry ice and freeze-thaw 3 cycles with 37 oC warm water.

4. Use '''blunted needle to break&nbsp; sticky DNA'''. Spin at maximum speed for 5 min using a microcentrifuge.

5. Transfer supernantant solution to another tube. This is the soluble fraction ('''S''')

6. Add 0.5 ml&nbsp;'''Lysis Buffer P to pellet''':

'''Lysis Buffer P:&nbsp;'''10 mM Imidozale, 20 mM Tris-HCl pH 8.0, 500&nbsp;NaCl, &nbsp;0.5% SDS,&nbsp;'''4 M Urea or 4 M'''

7. Use blunted needle to break &nbsp;pellet. Spin at maximum speed for 10 min using a microcentrifuge. Collect supernatant into another tube. This is the solubilized fraction from pellet ('''IB''').



8. Add 50 ul regenerated Ni beads (50% suspension) to both S & IB fractions.

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

10. Spin down beads gently (1000 rpm, 1 min). Transfer supernatant into the original tube.

11. Wash beads 3 times with 500 ul of &nbsp;'''Washing Buffer''':&nbsp;<u>10 mM Imidozale</u>,&nbsp; 20 mM Tris-HCl pH 8.0, 500 mM NaCl&nbsp;&nbsp;<u>4M Urea</u>, &nbsp;0.1% SDS,&nbsp;&nbsp;Spin down beads gently at 1000 rpm for 1 min, carefully remove washing solutions (do not pipette out beads).



12. Add 20 ~ 30 ul&nbsp;'''Elution Buffer (Buffer B):'''&nbsp;<u>150 mM Imidozale</u>, 20 mM Tris-HCl, pH 8.0. Incubate at RT for at least 1 min.

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

14. 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, label bottle with a tape: Used Ni Beads)

15. Run SDS-PAGE.

16. Stain with SimplyBlue or other protein dyes.

17. Destain with water

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

19.. Make a glycerol stock of expression clones.