Creating Competent E. coli Cells: Difference between revisions
Jump to navigation
Jump to search
PreDec2022>EricMiller No edit summary |
EricMiller (talk | contribs) No edit summary |
||
(6 intermediate revisions by 3 users not shown) | |||
Line 10: | Line 10: | ||
===Special Reagents Needed=== | ===Special Reagents Needed=== | ||
::*TF1 Solution | ::*TF1 Solution — Add in this order | ||
:::# | :::# 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) | ||
:::# | :::# 4.5mL 1M Potassium Acetate | ||
:::# | :::# 15mL 1M KCl | ||
:::# | :::# 3 mL 0.5M CaCl2 | ||
:::# Fill to | :::# 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. | ||
:::# Filter sterilize only. Place in 3rd floor cold room. | :::# Filter sterilize only. Place in 3rd floor cold room. | ||
::* TF2 solution. Place in the 3rd floor cold room. | ::* 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 | :::# 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. | :::# Filter sterilize only. Place in 3rd floor cold room. | ||
::* Growth media | ::* 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 | :::# Fill a graduated cylinder to ~700ml with MilliQ water | ||
::# Add 4g Yeast Extract | :::# Add 4g Yeast Extract | ||
::# Add 16g Bactotryptone | :::# Add 16g Bactotryptone | ||
::# Add 4g MgSO4 | :::# Add 4g MgSO4 | ||
::# Dissolve and fill up to 800ml with water | :::# Dissolve and fill up to 800ml with water | ||
::# Adjust to pH 7.6 with KOH (not NaOH) | :::# Adjust to pH 7.6 with 1M KOH (not NaOH) | ||
::# Put 400ml in each of two 2L flasks and immediately autoclave. | :::# Put 400ml in each of two 2L flasks and immediately autoclave. | ||
===Day 1=== | ===Day 1=== | ||
Line 53: | Line 53: | ||
'''Important:''' Treat cells gently after addition of TF1 Solution — no vortexing or vigorous pipetting — as this will greatly reduce the competency. | '''Important:''' Treat cells gently after addition of TF1 Solution — no vortexing or vigorous pipetting — as this will greatly reduce the competency. | ||
# Inoculate both 400ml LB flasks with 4.5ml of overnight growth and shake at 37 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 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 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. | # 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 | # Place flasks on ice, or even better, in an ice bath. Take to Superlab. | ||
# Put cells into four total large ultra-centrifuge tubes, and balance to 0.1g. This will be ~200ml 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 ultra-centrifuge. | # Pellet cells for 20 minutes at 3700 rpm / 3200 x g at 4 degrees in Superlab ultra-centrifuge. | ||
# Decant (gently pour off) supernatant and gently resuspend cells in | # 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 two 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 and the electronic pipettor. | # 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
- Do you know how to triple-streak cells onto a plate?
- Do you have the necessary media plates (LB, Tryptic Soy, etc.)?
- 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
- 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)
- 4.5mL 1M Potassium Acetate
- 15mL 1M KCl
- 3 mL 0.5M CaCl2
- 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.
- Filter sterilize only. Place in 3rd floor cold room.
- 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.
- 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
- Add 4g Yeast Extract
- Add 16g Bactotryptone
- Add 4g MgSO4
- Dissolve and fill up to 800ml with water
- Adjust to pH 7.6 with 1M KOH (not NaOH)
- Put 400ml in each of two 2L flasks and immediately autoclave.
Day 1
- Triple-streak previous competent cells for single colonies. Use a Tryptic Soy plate or an LB plate with antibiotics, if applicable.
- Create TF1 solution
- Create TF2 solution
- Create Growth media
- Create 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.
- Place two 50ml Falcon tubes in the 3rd floor cold room.
Day 2
- 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.
- 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.
- 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 to Superlab.
- 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 ultra-centrifuge.
- 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 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.
- 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 and the electronic pipettor.
- Flash freeze aliquots in liquid nitrogen.