Crm at 15:34, 10 October 2018

2018-10-10T15:34:01Z

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== Transformation and Small Scale Expression Screening ==

1. If a clone in a vector has 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  is ~0.8~1, add 1 M IPTG to final concentration of  0.2~0.5 mM. induce 4 hours at 37 oC or 25 ~30 oC o/m.

2. If there is no expression bacteria for the  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 '''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, , when OD600  is ~0.8~1, add 1 M IPTG to final concentration of  0.2~0.5 mM. induce 4 hours at 37 oC or 25 ~30 oC o/m.

3. Transfer cell culture to   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  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 '''Lysis Buffer P to pellet''':

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

7. Use blunted needle to break  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  '''Washing Buffer''': 10 mM Imidozale,  20 mM Tris-HCl pH 8.0, 500 mM NaCl  4M Urea,  0.1% SDS,  Spin down beads gently at 1000 rpm for 1 min, carefully remove washing solutions (do not pipette out beads).

12. Add 20 ~ 30 ul '''Elution Buffer (Buffer B):''' 150 mM Imidozale, 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 to a row of PCR tubes (reuse, do not throw away).  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.

== 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,  grow them in '''5 ml or 15 ml''' 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  or  [[Phosphate_Buffer_Chart|50 mM Na-Phosphate, pH 7.4]], 300  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 

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

Steps 11 and 12 are optional:

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

'''Lysis Buffer P''': 10 mM Imidozale, 20 mM Tris-HCl pH 8.0, 600 NaCl,   [[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  '''Washing Buffer''': 35 mM Imidozale, <strike>20 mM Tris-HCl pH 8.0, 600 mM NaCl</strike>, [[Phosphate_Buffer_Chart|50 mM Na-Phosphate, pH 7.4]], 300 mM NaCl, 4M Urea, 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  '''Washing Buffer II''': 35 mM Imidozale,   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:''' 300 mM Imidozale, 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).  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.

== pT7, pET,  pT7A3, pT7A30, pMal, pBAD Screening ==

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

2. Grow on an agar plate with proper selection antibiotics.

3. Pick a few colonies,  grow them in '''2'''~4 or 15 ml  LB/antibiotics (optionally, 0.5% Glucose, Glucose can suppress leaky/basal expression) in a autoclaved glass tube, at 37 oC for 4~6 hours until OD600 is 0.8~1.5.

4.  Transfer 100 ul culture into a microcentrifuge tube with 0.5 ml  LB/antibiotics. Add 2 ml LB/antibiotic medium, grow up uninduced cells at 37 oC for making glycerol stock.

5. Add 1 M IPTG to the glass tube to final conc of  01.~ '''0.5 mM IPTG. Induce ''' for 1 to '''4 hours''' at '''37 oC'''., or 25~30 oC overnight
<blockquote>Other optimization conditions: pT7,  pET,   pMAL vectors: 0.1 to 1 mM IPTG at 37 oC for 4 hours, or 25~30 oC o/n. </blockquote><blockquote>pT7A3 and pT7A30: 0.1 mM IPTG 25~30 oC o/n pBad: 0.02~0.2% arabinose </blockquote><blockquote>Use Rosetta2 BL21 (DE3)/pLysS or BL21 Star (DE3)/pRare2pLysS for human or mouse genes, or other competent cells transformed with pRare2pLysS. </blockquote><blockquote>Use Nico21(DE3)/pLysN for genes cloned from E coli or phage. </blockquote><blockquote>Use C43(DE3)/pRare2pLysS or C43(DE3)/pLysN for toxic proteins</blockquote>
7. Spin down cells.

8. Add 400 ul of Lysis '''Buffer S''' to induced samples. Votex to suspend cells. Transfer the suspension to microcentrifuge tube by pouring.

'''Lysis Buffer S''': 10 mM Imidozale, <strike>20 mM Tris-HCl pH 8.0, 600 mM NaCl</strike>, [[Phosphate_Buffer_Chart|50 mM Na-Phosphate, pH 7.4]], 300 mM NaCl, 1 mM beta-ME, 0.1% Triton X-100, <strike>10% glycerol</strike>.

9. Freeze-thaw cells 1 to 3 cycles. If lysis does not occure, add 2 ul 100X lysozyme, incubate at 37 oC for 15~30 min until the cells are lysed. If the lysate is too sticky (too much DNA), add 1 ul Benzonase. Incubate at 37 oC for 15 min, or use needle to break DNA

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

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

'''Lysis Buffer P''': 20 mM Imidozale, <strike>20 mM Tris-HCl pH 8.0, 600 mM NaCl</strike>, [[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'''

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

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

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

15. Spin down beads gently (1000 rpm, 1 min).

16. Wash beads 3 times with 200 ul of  '''Washing Buffer''': '''20''' mM Imidozale, <strike>20 mM Tris-HCl pH 8.0, 600 mM NaCl</strike>, [[Phosphate_Buffer_Chart|50 mM Na-Phosphate, pH 7.4]], 300 mM NaCl, 4M Urea, 1 mM beta-ME, 0.1% SDS, <strike>0.1% Triton X-100</strike>, <strike>10% glycerol</strike>. Spin down bead 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 Washing Buffer: 35 mM Imidozale, 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:''' 300 mM Imidozale, 20 mM Tris-HCl pH 8.0. Incubate at RT for at least 1 min.

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

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

21. Run SDS-PAGE.

22. Stain with SimplyBlue or other protein dye.

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. --[[User:Paul.xu|Paul.xu]] 22:38, 1 April 2013 (EST)
[[Category:Protocols]]

Generic Protein Expression Screening Protocol - Revision history

Crm at 15:34, 10 October 2018