Creating Competent E. coli Cells: Difference between revisions

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==Goal==
==Goal==
*Goal: To create competent E. coli cells using either of the two methods listed below.
*Goal: To create competent <em>E. coli</em> cells using either of the two methods listed below.


==Pre-Protocol Questions==
==Pre-Protocol Questions==
#Do you know how to triple-streak cells onto a plate?
#Do you know how to triple-streak cells onto a plate?
#Do you have the necessary media plates (LB, Tryptic Soy, etc.)?
#Do you have the necessary media plates (LB, Tryptic Soy, etc.)?
#Do you know how to use the centrifuge?
#Do you know how to use the huge ultra-centrifuge in Superlab?


==Version 1: Traditional CaCl2 competence==
==Current Version: CaCl2 Competent Cells without Sodium Ions (from Bash)==


===Day 1===
===Special Reagents Needed===
 
::*TF1 Solution — Add in this order
# Triple-streak previous competent cells for single colonies. Use a Tryptic Soy plate or an LB plate with antibiotics, if applicable.
:::# 28.1 mL 80% glycerol
 
:::# 7.5 mL 1M MnCl2 in acidified water (83.3ul concentrated HCl added to 5ml water; add 1.26g MnCl2 and dissolve; add water up to 10ml. If the solution has a brown-orange precipitate, then MnCl2 oxidized and a new solution is needed)
===Day 2===
:::# 4.5mL 1M Potassium Acetate
 
:::# 15mL 1M KCl
# From this grown plate, pick a single colony and inoculate 10ml LB (+ antibiotics if applicable) in a 50ml Erlenmeyer flask. Grown overnight at 37 degrees with shaking.
:::# 3 mL 0.5M CaCl2
::a. 37 degree shaking incubator located next to the two fridges.
:::# Fill to 150 mL and adjust pH to 5.8 with 0.2M acetic acid (or 0.2M KOH). Go very slow with this step; if the solution goes above 7.0, then the MnCl2 will precipitate and will not go back into solution.
# Have a 2L Erlenmeyer flask with 500mL LB autoclaved and ready to go for tomorrow.  
:::# Filter sterilize only. Place in 3rd floor cold room.
# Have three 250ml centrifuge tubes autoclaved and ready to go. Ask Eric where these are.
# Autoclave 50ml of 0.1M CaCl2 in 15% glycerol (40.7ml 0.1M CaCl2 + 9.3ml of 80% glycerol)
# Label and set up 100 0.6ml tubes for the eventual aliquots.


===Day 3===
::* TF2 solution. Place in the 3rd floor cold room.
:::# 200 ul 1M MOPS, pH 6.5
:::# 200ul mL 1M KCl
:::# 3 mL 0.5M CaCl2
:::# 3.75 mL 80% glycerol
:::# Fill to 20mL with water and adjust pH to 6.5 with 0.2M acetic acid or KOH
:::# Filter sterilize only. Place in 3rd floor cold room.


        '''Important:''' Treat cells gently after addition of CaCl2 — no vortexing or vigorous pipetting — as this will greatly reduce the competency.
::* Growth media (Either for 800ml of cells, or double the number of flasks. There will be enough TF1 and TF2 for this double volume.)
 
:::# Fill a graduated cylinder to ~700ml with MilliQ water
# Transfer 5ml of this overnight culture into the 2L flask containing 500ml LB. Grow at 37 degrees with shaking.
:::# Add 4g Yeast Extract
# Immediately after transferring this culture, sterile syringe filter (0.22um filter) the 50ml 0.1M CaCl2 in glycerol. Put on ice and place at 4 degrees. Additionally, chill two 50ml plastic tubes.
:::# Add 16g Bactotryptone
# Grow culture until OD600 is ~0.4. (Use spectrophotometer to check the OD.) This will take 2-3 hours.
:::# Add 4g MgSO4
# From now on, everything must be on ice!
:::# Dissolve and fill up to 800ml with water
# Roughly divide the 500ml of grown E. coli into the three 250ml centrifuge tubes. Do not be tempted to only use 2 tubes; this will result in LB spilling out into the centrifuge.
:::# Adjust to pH 7.6 with 1M KOH (not NaOH)
# Put these tubes in a styrofoam box containing ice; place in the refrigerator. These need to incubate for a total of 20 minutes.
:::# Put 400ml in each of two 2L flasks and immediately autoclave.
# Go to Superlab and turn on the large centrifuge. Ensure that it is set to 4 degrees and that it is actively decreasing in temperature.
# Also, turn on the tabletop swinging bucket centrifuge in Superlab. Ensure that it is set to 4 degrees and that it is actively decreasing in temperature.
# After the 20-minute incubation at 4 degrees, weigh out the three centrifuge tubes and balance them to the nearest 0.5g. This will take some time; keep the E. coli on ice.
# Centrifuge the cultures at 3000 x g at 4 degrees for 10 minutes.
# Turn off centrifuge. Take cultures back to the MEE lab and slowly pour off supernatant into a large graduated beaker.
# Resuspend the cells gently in 30ml (total) ice-cold 0.1M CaCl2 + glycerol. Transfer to the two pre-chilled 50ml tubes.
# Incubate cells on ice for 30 minutes at 4 degrees.
# After the 30-minute incubation at 4 degrees, weigh out the two 50ml tubes and balance them to the nearest 0.5g.
# Centrifuge for 5 minutes at 3000 x g at 4 degrees using the swinging bucket centrifuge in Superlab.
# Turn off centrifuge. Take cultures back to the MEE lab and slowly pour off supernatant into same large graduated beaker as before. When you have time, add enough Virkon to this to kill off any cells and dispose of.
# Resuspend cells gently with 8ml total ice-cold 0.1M CaCl2 in glycerol. Do not vortex!
# Transfer 260ul aliquots into 0.6ml tubes; flash-freeze in liquid nitrogen and store at -80 degrees. Use the electronic pipettor to do this faster!
 
==Version 2, from Bash: More complicated, but with more success==


===Day 1===
===Day 1===


# Triple-streak previous competent cells for single colonies. Use a Tryptic Soy plate or an LB plate with antibiotics, if applicable.
# Triple-streak previous competent cells for single colonies. Use a Tryptic Soy plate or an LB plate with antibiotics, if applicable.
# Do we have the following items? If not, make them.
# Create TF1 solution
::* ~200 0.6ml tubes labeled with a color not currently used by other competent cells. Place in the 3rd floor cold room.
# Create TF2 solution
::* 1 empty microcentrifuge rack. Place in the 3rd floor cold room.
# Create Growth media
::* TF1 solution. Place in the 3rd floor cold room.
# Create p1000 tips with 0.25cm cut off sterilely. Place in the 3rd floor cold room.
# 3mL 1M Potassium Acetate
# Sterilized large ultra-centrifuge tubes. Place in the 3rd floor cold room.
# 10mL 1M KCl
# Place two 50ml Falcon tubes in the 3rd floor cold room.
# 2 mL 0.5M CaCl2
# 5 mL 1M MnCl2
# 18.75 mL 80% glycerol
# Fill to 100 mL and adjust pH to 5.8 with 0.2M acetic acid or NaOH. Solution will change to slight brownish yellow color.
# Filter sterilize only.
* TF2 solution. Place in the 3rd floor cold room.
# 1 mL MOPS, pH 6.5
# 1 mL 1M KCl
# 15 mL 0.5M CaCl2
# 18.75 mL 80% glycerol
# Fill to 100mL with water and adjust pH to 6.5
# Filter sterilize only.
* p1000 tips with 0.25cm cut off sterilely. Place in the 3rd floor cold room.
* Sterilized large ultra-centrifuge tubes. Place in the 3rd floor cold room.
* 2 x 50ml Falcon tubes. Place in the 3rd floor cold room.
* 2 x 2L flasks with 400ml media in each flask (Make media in flasks and immediately autoclave).
# 2g Yeast Extract
# 8g Bactotryptone
# 2g MgSO4
# Fill to 400ml and adjust to pH 7.6 with KOH (not NaOH)


===Day 2===
===Day 2===
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===Day 3===
===Day 3===
# Inoculate both 500ml LB flasks with 4.5ml of overnight growth and shake at 37 degrees.
'''Important:''' Treat cells gently after addition of TF1 Solution — no vortexing or vigorous pipetting — as this will greatly reduce the competency.
# Turn on the Superlab centrifuge, and chill to 4 degrees.
 
# Inoculate both 400ml LB flasks with 4.5ml of overnight growth and shake at 37 degrees. Use 2ml if you are using four 400ml LB flasks.
# Turn on the Superlab ultra-centrifuge, and chill to 4 degrees.
# Turn on Superlab swinging rotor centrifuge, and chill to 4 degrees.
# Turn on Superlab swinging rotor centrifuge, and chill to 4 degrees.
# Measure OD every hour and every 30 minutes, until O.D. at 600nm is 0.2-0.4 (~3 hours). Using a 13mm tube, this translates to Ab 0.25-0.52.
# Measure OD after 1 hour and then every 30 minutes, until O.D. at 600nm is 0.2-0.4 (~3 hours). Using a 13mm tube, this translates to Ab 0.25-0.52.
# Place flasks on ice, or even better, in an ice bath. Take up to 3rd floor cold room.
# Place flasks on ice, or even better, in an ice bath. Take to Superlab.
# Put cells into large ultra-centrifuge tubes, and balance to 0.1g. ~250ml each.
# Put cells into four total large ultra-centrifuge tubes, and balance to 0.1g. This will be ~200ml each. (Use six ultra-centrifuge tubes, each with ~266ml, if you are using four 400ml LB flasks.)
# Pellet cells for 20 minutes at 3700 rpm / 3200 x g at 4 degrees in Superlab centrifuge.
# Pellet cells for 20 minutes at 3700 rpm / 3200 x g at 4 degrees in Superlab ultra-centrifuge.
# Decant supernatant and gently resuspend cells in 40ml ice cold TF1 solution per ultra-centrifuge tube by pipetting up and down with a serological pipette. Keep on ice.
# Decant (gently pour off) supernatant and gently resuspend cells in 20ml ice cold TF1 solution per ultra-centrifuge tube by pipetting up and down with a serological pipette. Keep on ice.
# Consolidate cells to 2 x 50ml Falcon tubes and chill on ice for 20 minutes. Balance to 0.1g.
# Consolidate cells to two 50ml Falcon tubes and chill on ice for 20 minutes. Balance to 0.1g. (Use three 50ml Falcon tubes if you are using four 400ml LB flasks.)
# Pellet cells for 20 minutes at 3700 rpm at 4 degrees in the swinging rotor centrifuge.
# Pellet cells for 20 minutes at 3700 rpm at 4 degrees in the swinging rotor centrifuge.
# Decant supernatant and gently resuspend cells in 2ml ice cold TF2 solution per pellet by pipetting up and down with a cut-off p1000 pipette tip. Chill on ice for 10 minutes. Consolidate into one tube.
# Decant supernatant and gently resuspend cells in 2ml ice cold TF2 solution per pellet by pipetting up and down with a cut-off p1000 pipette tip. Chill on ice for 10 minutes. Consolidate into one tube.
# In cold room, aliquot 50 ul into the 0.6ml tubes, using the cut-off p1000 tips.
# In cold room, aliquot 50 ul into the 0.6ml tubes, using the cut-off p1000 tips and the electronic pipettor.
# Flash freeze aliquots in liquid nitrogen.
# Flash freeze aliquots in liquid nitrogen.

Latest revision as of 13:40, 10 June 2024

Goal

  • Goal: To create competent E. coli cells using either of the two methods listed below.

Pre-Protocol Questions

  1. Do you know how to triple-streak cells onto a plate?
  2. Do you have the necessary media plates (LB, Tryptic Soy, etc.)?
  3. Do you know how to use the huge ultra-centrifuge in Superlab?

Current Version: CaCl2 Competent Cells without Sodium Ions (from Bash)

Special Reagents Needed

  • TF1 Solution — Add in this order
  1. 28.1 mL 80% glycerol
  2. 7.5 mL 1M MnCl2 in acidified water (83.3ul concentrated HCl added to 5ml water; add 1.26g MnCl2 and dissolve; add water up to 10ml. If the solution has a brown-orange precipitate, then MnCl2 oxidized and a new solution is needed)
  3. 4.5mL 1M Potassium Acetate
  4. 15mL 1M KCl
  5. 3 mL 0.5M CaCl2
  6. Fill to 150 mL and adjust pH to 5.8 with 0.2M acetic acid (or 0.2M KOH). Go very slow with this step; if the solution goes above 7.0, then the MnCl2 will precipitate and will not go back into solution.
  7. Filter sterilize only. Place in 3rd floor cold room.
  • TF2 solution. Place in the 3rd floor cold room.
  1. 200 ul 1M MOPS, pH 6.5
  2. 200ul mL 1M KCl
  3. 3 mL 0.5M CaCl2
  4. 3.75 mL 80% glycerol
  5. Fill to 20mL with water and adjust pH to 6.5 with 0.2M acetic acid or KOH
  6. Filter sterilize only. Place in 3rd floor cold room.
  • Growth media (Either for 800ml of cells, or double the number of flasks. There will be enough TF1 and TF2 for this double volume.)
  1. Fill a graduated cylinder to ~700ml with MilliQ water
  2. Add 4g Yeast Extract
  3. Add 16g Bactotryptone
  4. Add 4g MgSO4
  5. Dissolve and fill up to 800ml with water
  6. Adjust to pH 7.6 with 1M KOH (not NaOH)
  7. Put 400ml in each of two 2L flasks and immediately autoclave.

Day 1

  1. Triple-streak previous competent cells for single colonies. Use a Tryptic Soy plate or an LB plate with antibiotics, if applicable.
  2. Create TF1 solution
  3. Create TF2 solution
  4. Create Growth media
  5. Create p1000 tips with 0.25cm cut off sterilely. Place in the 3rd floor cold room.
  6. Sterilized large ultra-centrifuge tubes. Place in the 3rd floor cold room.
  7. Place two 50ml Falcon tubes in the 3rd floor cold room.

Day 2

  1. From this grown plate, pick a single colony and inoculate 10ml LB (+ antibiotics if applicable) in a 50ml Erlenmeyer flask. Grown overnight at 37 degrees with shaking.

Day 3

Important: Treat cells gently after addition of TF1 Solution — no vortexing or vigorous pipetting — as this will greatly reduce the competency.
  1. Inoculate both 400ml LB flasks with 4.5ml of overnight growth and shake at 37 degrees. Use 2ml if you are using four 400ml LB flasks.
  2. Turn on the Superlab ultra-centrifuge, and chill to 4 degrees.
  3. Turn on Superlab swinging rotor centrifuge, and chill to 4 degrees.
  4. Measure OD after 1 hour and then every 30 minutes, until O.D. at 600nm is 0.2-0.4 (~3 hours). Using a 13mm tube, this translates to Ab 0.25-0.52.
  5. Place flasks on ice, or even better, in an ice bath. Take to Superlab.
  6. Put cells into four total large ultra-centrifuge tubes, and balance to 0.1g. This will be ~200ml each. (Use six ultra-centrifuge tubes, each with ~266ml, if you are using four 400ml LB flasks.)
  7. Pellet cells for 20 minutes at 3700 rpm / 3200 x g at 4 degrees in Superlab ultra-centrifuge.
  8. Decant (gently pour off) supernatant and gently resuspend cells in 20ml ice cold TF1 solution per ultra-centrifuge tube by pipetting up and down with a serological pipette. Keep on ice.
  9. Consolidate cells to two 50ml Falcon tubes and chill on ice for 20 minutes. Balance to 0.1g. (Use three 50ml Falcon tubes if you are using four 400ml LB flasks.)
  10. Pellet cells for 20 minutes at 3700 rpm at 4 degrees in the swinging rotor centrifuge.
  11. Decant supernatant and gently resuspend cells in 2ml ice cold TF2 solution per pellet by pipetting up and down with a cut-off p1000 pipette tip. Chill on ice for 10 minutes. Consolidate into one tube.
  12. In cold room, aliquot 50 ul into the 0.6ml tubes, using the cut-off p1000 tips and the electronic pipettor.
  13. Flash freeze aliquots in liquid nitrogen.
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