Protocols

General Protocols

LB Broth (Miller)

Composition per liter

• 10 g casein peptone
• 5 g yeast extract
• 10 g sodium chloride

Directions: Dissolve 25 g total in 1 L purified water. Heat and agitate until completely dissolved. Sterilize by autoclaving for 15 min.

LB Plates

Add 15 g agar per 1 L LB broth media. Completely dissolve with heat and stirring before autoclaving to avoid undissolved pockets that prevent even dispersion.

Difco MRS Lactobacilli Broth (BD)

Composition per liter

• 10 g proteose peptone no. 3
• 10 g beef extract
• 5 g yeast extract
• 20 g dextrose
• 1 g polysorbate 80
• 2 g ammonium citrate
• 5 g sodium acetate
• 0.1 g magnesium sulfate
• 0.05 g manganese sulfate
• 2 g dipotassium phosphate

Directions: Suspend 55 g powder in 1 L purified water. Mix thoroughly. Heat with frequent agitation and boil 1 min to fully dissolve. Autoclave at 121°C for 15 min.

MRS Lactobacillus Agar Plates

Add 15 g agar per 1 L MRS broth. Dissolve completely with heat and stirring before autoclaving to ensure uniform dispersion.

50% Glycerol

• 50 mL 100% glycerol
• 50 mL Milli‑Q water

Autoclave to sterilize. Use 1:1 with bacterial culture to make glycerol stocks.

Materials for Competent Cell Creation

• 1 M KCl
• 1 M Glucose
• 1 M MgCl₂ (hexahydrate)
• 1 M MOPS
• 0.1 M Acetic acid
• 1 M Potassium hydroxide

Bacterial Transformation

AddGene: Heat‑Shock Protocol

1. Take competent cells out of −80°C and thaw on ice (approximately 20–30 mins).
2. Remove agar plates (containing the appropriate antibiotic) from 4°C and let warm to room temperature; optionally pre‑incubate at 37°C.
3. Mix 1–5 μL DNA (usually 10 pg–100 ng) into 20–50 μL competent cells in a microcentrifuge or Falcon tube. GENTLY mix by flicking the tube a few times.

Pro‑Tip: Transformation efficiencies are ~10‑fold lower for ligation products than for intact control plasmids.

4. Incubate the competent cell/DNA mixture on ice for 20–30 mins.
5. Heat shock by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30–60 secs (45 secs is usually ideal; varies by cells).
6. Return tubes to ice for 2 min.
7. Add 250–1,000 μL LB or SOC (no antibiotic) and grow in a 37°C shaking incubator for 45 min.

Pro‑Tip: Outgrowth allows expression of antibiotic‑resistance genes. Less critical for Ampicillin; more important for other antibiotics.

8. Plate some or all of the transformation onto a 10 cm LB agar plate containing the appropriate antibiotic.

Pro‑Tips: Plate 50 μL on one plate and the rest on a second to get single colonies and recover all transformants. If culture volume is too big, gently pellet and resuspend to reduce liquid on plates. Ensure plates are dry before growth to avoid bacterial diffusion.

9. Incubate plates at 37°C overnight.

Heat-Shock Protocol

Source: AddGene – Bacterial Transformation

Heat Shock Protocol Obtained from Catherine Vaerewyck

Used for: Inserting plasmids into strains without the rigors of electro shock

Materials: 60 mM CaCl₂·2H₂O for Heat Shock

1. Weigh 2.21 g CaCl₂·2H₂O into a clean mixing bottle.
2. Add 25 mL TRIS pH 7.5.
3. Weigh 37.5 g of 100% glycerol.
4. Measure 250 mL nanopure water and use it to wash glycerol from the weigh boat into the bottle.
5. If needed, bring to 250 mL with nanopure water.
6. Shake to mix, then filter‑sterilize.

Method

1. Prepare electrocompetent cells as follows:
   1. Place CaCl₂ solution, plasmid‑receiving cells, and tubes on ice; keep all other supplies on ice.
   2. Let components sit ~30 min; simultaneously pre‑warm LB tubes and antibiotic plates at 37°C.
   3. Divide plasmid‑receiving cells between four chilled microcentrifuge tubes.
   4. Spin 9,000 rpm for 2 min.
   5. Decant supernatant; resuspend each pellet in 1 mL chilled CaCl₂ solution.
   6. Spin 9,000 rpm for 2 min.
   7. Decant supernatant.
   8. Resuspend one pellet in 1 mL CaCl₂, then use that same milliliter to resuspend the other pellets into a single tube (1 mL for four pellets).
   9. Spin combined tube 9,000 rpm for 2 min.
   10. Decant supernatant; resuspend pellet in 100 μL CaCl₂.
2. Prepare purified plasmid as outlined in “Plasma Prep”.
3. Pre‑warm an LB tube at 37°C.
4. Combine 100 μL electrocompetent cells and 4 μL purified plasmid; gently mix; incubate on ice 30 min.
5. Heat block 42°C for 90 s.
6. Return to ice 2 min.
7. Bring volume to 1 mL by adding 950 μL pre‑warmed LB.
8. Incubate 37°C for 1 h.
9. Plate as outlined in Electroporation steps 15–19.
10. Incubate plates at 37°C overnight.

Additional Resources / Sources – P. aeruginosa

• https://pubmed.ncbi.nlm.nih.gov/9661666/
• https://pubmed.ncbi.nlm.nih.gov/15987659/
• https://www.nature.com/articles/nprot.2015.115

Chemical Competent E. coli Transformations – Protocol by Owen ‘Gus’ Collars

1. Thaw RbCl chemically competent E. coli on ice.
2. Add desired amount of DNA (generally 10–20 ng) to 30–50 μL of E. coli (depends on density).
3. Incubate on ice for 30 minutes.
4. Heat shock at 42°C for 30 seconds.
5. Incubate on ice for 1 minute.
6. Add 300 μL LB.
7. Recover 1 hour at 37°C.
8. Spin 13,000 rpm for 1 minute; resuspend in ~30 μL and plate on selective media.

Bacterial Transformation with Chemical Competent Cells – NEB (via Kurt Kohler)

1. Thaw competent cells on ice.
2. Chill ~5 ng (2 μL) of ligation mixture in a 1.5 mL tube.
3. Add 50 μL competent cells to DNA. Mix gently by pipetting or flicking 4–5×. Do not vortex.
4. Place on ice 30 minutes. Do not mix.
5. Heat shock at 42°C for 30 seconds*. Do not mix.
6. Add 950 μL room‑temperature media*.
7. Incubate 37°C for 60 minutes. Shake 250 rpm or rotate.
8. Warm selection plates to 37°C.
9. Spread 50–100 μL of cells/ligation mixture onto plates.
10. Incubate overnight at 37°C.

* For heat‑shock duration/temperature and recovery media, follow the competent‑cell manufacturer’s recommendations.

Competent Cells

Chemical Competent Cells

Protocol adapted from the Hanahan RbCl₂ method by Allan Caplan at the University of Idaho. Graciously received protocol from Dr. Rebecca Prest of Dr. Patricia Champion’s Lab.

Introduction

Electrically competent cells are simpler to prepare but require more skill during transformation. Chemically competent cells take longer to prepare (especially the buffers) but are straightforward to transform.

Media and Buffers

SOC plus Mg

• Tryptone 20 g
• Yeast Extract 5 g
• 1 M KCl 2.5 mL
• 1 M Glucose 20 mL
• 1 M MgCl₂ 20 mL

Adjust pH to 7.0 with KOH (do not use NaOH). Bring to 1 L with H₂O. Filter‑sterilize with a 0.22 μm cup filter. Do not autoclave.

TFB I

Add H₂O to ~90 mL, adjust pH to 5.8 with 0.1 M acetic acid (poorly buffered). Bring to 100 mL with H₂O. Filter‑sterilize and store at 4°C for up to 1 year (discard if brown).

TFB II

Adjust pH to 6.5 with KOH (do not use NaOH). Bring to 150 mL with H₂O and filter‑sterilize. Store at 4°C for up to 1 year (discard if brown or bright‑yellow MOPS).

Procedure

1. Start an overnight from a single colony or glycerol stock in 2 mL LB at 37°C (14 mL round‑bottom tube).
2. Add 500 μL overnight to 50 mL SOC+Mg with antibiotics in a sterile 250 mL baffled flask. Grow 37°C for 2–3 h to A₆₀₀ 0.4–0.6.
3. Chill flask on ice 10–15 min (keep everything cold from here: tubes, tips, rotors, buffers).
4. Pre‑label 10–20 sterile tubes for aliquots; place in a freezer box (no lid) at −20°C.
5. Spin 50 mL culture 5 min at 5000 rpm, 4°C; decant; keep on ice.
6. Resuspend in 20 mL ice‑cold TFB I; never vortex.
7. Incubate on ice 5 min.
8. Spin 5 min at 5000 rpm, 4°C; decant.
9. Resuspend in 2 mL ice‑cold TFB II.
10. Incubate on ice 15–60 min.
11. Aliquot into pre‑chilled tubes (ideally in a cold room; a plastic rack pre‑chilled at −80°C helps).
12. Freeze aliquots in an open freezer box at −80°C for several hours.

Use like commercial chemical competent cells (thaw on ice → add DNA → 30 s 42°C heat shock → chill → recover 1 h in SOC at 37°C).

Electrocompetent L. plantarum

Reference protocol

Making electrocompetent cells

1. Dilute 10 mL overnight 1:10 into fresh MRS. Grow 30°C, 3–4 h to O.D.₆₀₀ ≈ 0.85.
2. Pellet (4°C); wash 2× with 10 mL chilled MgCl₂ (10 mM) and 1× with 10 mL 0.5 M sucrose + 10% glycerol.
3. Resuspend in 2–3 mL of the same solution and keep on ice ≤4 h (frozen cells lose 1–2 logs of competence).

Electroporation

1. Add 100 ng plasmid DNA (in 5 μL TE) to 50 μL (3×10⁸) competent cells; mix gently; transfer to a pre‑chilled cuvette.
2. Dry cuvette exterior; electroporate at 1,300 V; time constant 5 ms.
3. Immediately add 1 mL pre‑warmed MRS; recover 30°C, 3 h with shaking.
4. Plate on selective MRS and incubate 30°C for 48 h.

Expected: up to 5.8×10⁵ ± 2.2×10⁵ transformants/μg DNA.

Making Calcium Competent Cells for Transformation of E. coli

Protocol obtained from Kurt Kohler

Day 1

1. Streak a fresh LB plate (no antibiotic). Grow overnight 37°C.

Day 2

1. Autoclave: 1 L LB, 1 L 100 mM CaCl₂, 1 L 100 mM MgCl₂, 100 mL 85 mM CaCl₂ + 15% glycerol; 4 centrifuge bottles; microfuge tubes.
2. Chill overnight (4°C): CaCl₂, MgCl₂, 85 mM CaCl₂ + 15% glycerol, centrifuge rotor.
3. Start 10 mL LB culture from single colony (no antibiotics). Grow overnight 37°C, shaking.

Notes: You may substitute SOB/2×YT. Ensure glassware is detergent‑free.

Day 3

1. Inoculate 1 L LB with 10 mL starter. Monitor OD₆₀₀; when 0.35–0.4, chill immediately.
2. Keep all steps at 4°C hereafter; pre‑chill everything.
3. (Spin #1) Split into 4×250 mL; spin 3000 g, 15 min, 4°C.
4. Resuspend each pellet in ~100 mL cold MgCl₂; combine into one bottle.
5. (Spin #2) 2000 g, 15 min, 4°C.
6. Resuspend in ~200 mL cold CaCl₂; incubate on ice ≥20 min.
7. (Spin #3) 2000 g, 15 min, 4°C. Chill a 50 mL conical.
8. Resuspend in ~50 mL 85 mM CaCl₂, 15% glycerol; transfer to the 50 mL tube.
9. (Spin #4) 1000 g, 15 min, 4°C.
10. Resuspend in 2 mL 85 mM CaCl₂, 15% glycerol (final OD₆₀₀ ≈ 200–250). Aliquot 50 μL and snap‑freeze.

Making Competent Treated Gram‑Positive Bacteria

From Bae JMB (2002) 315, 995–1007; edited by Z. Cusumano (1/20/16). Provided by K. Kohler.

Lysozyme buffer

• 10 mM Tris pH 8.0
• 25% sucrose
• 10 mM EDTA
• 50 mM NaCl

Electroporation solution

• 0.5 M sucrose — 17.1 g
• 10% glycerol — 10 mL
• H₂O to 100 mL final, filter‑sterilize (optionally pH to 7.0 with 10 μL of 12.5 M NaOH)

1. Grow cells in BHI overnight (20–50 mL). Back‑dilute 1:10 into 200–500 mL fresh BHI; grow 2.5 h to OD₆₀₀ 0.5–1.0.
2. Pellet; resuspend in lysozyme buffer (20–50 mL). Add lysozyme to 25 μg/mL; incubate 20 min at 37°C with rotation.
3. Wash 3× with ice‑cold electroporation solution; resuspend in 10–25 mL electroporation solution. Freeze −80°C in 200 μL aliquots.
4. Electroporate 5–10 μL plasmid in 0.2 cm cuvettes: 2.5 kV, 25 μF, 200 Ω, time constant ~4.9.
5. Recover per plasmid type: stable plasmids (ice 30′ → +1 mL media → 37°C 60′ → plate); temperature‑sensitive plasmids (ice 30′ → 30°C 60′ → plate with appropriate antibiotic).
6. Incubate 30°C for 2 days or at RT for 3 days.

Protocol volumes

Preparation of Electrocompetent Cells

Protocol originated by Michael McConnell (01/25/2019). ~500 μL output (≈5 transformations); scalable.

Materials

• Bacterial strain; selective agar media
• LB broth; autoclaved distilled water (1 L); autoclaved 10% glycerol
• Electroporation cuvettes (Bio‑Rad 1 mm or 2 mm)
• Plasmid DNA; LB/antibiotic plates

Protocol

Day 1

1. Streak plate; incubate 37°C overnight.
2. Prepare autoclaved water, 10% glycerol, LB, and antibiotic plates. Store water/glycerol at 4°C.

Day 2

1. Inoculate 100 mL LB with 3–4 colonies in a 250 mL flask.
2. Shake 180 rpm at 37°C to OD₆₀₀ ≈ 0.8 (3–4 h). Chill 30 min on ice (all further steps on ice).
3. Spin 5000×g, 7 min, 4°C; wash pellets 3× with 50 mL ice‑cold water.
4. Resuspend in 13 mL 10% glycerol; spin; resuspend pellet in 200 μL 10% glycerol.
5. Aliquot 100 μL into pre‑chilled tubes (freeze at −80°C for storage).
6. Transform ≤10 μL DNA; electroporate (Bio‑Rad Gene Pulser: Ec1 for 1 mm, Ec2 for 2 mm cuvettes).
7. Recover in 900 μL LB, 37°C, 180 rpm, 1 h; plate 100 μL on selective media; incubate overnight.

Making Your Own Electrocompetent Cells (Pilar Pérez Romero)

Before Starting

• Prepare sterile 10% glycerol for washes (≈2× culture volume).
• Incubate 1 colony in 10 mL LB for 16–18 h at 37°C, 250 rpm.
• Pre‑warm two 1 L flasks with 250 mL LB each.
• Add two drops of overnight to each flask; grow 37°C/250 rpm to OD₆₀₀ 0.5–0.7.
• Pre‑cool rotor to 4°C; place 1 L of 10% glycerol on ice.

Harvest and Wash

1. Ice‑chill cultures 15 min (keep ice‑cold thereafter).
2. Spin 5000 rpm, 10 min; discard supernatant.
3. Add 250 mL 10% glycerol to each bottle; fully resuspend by pipetting; spin 5000 rpm, 10 min.
4. Discard supernatant; add 250 mL 10% glycerol; resuspend completely.
5. Discard supernatant; resuspend cells in residual glycerol.

Freeze or Use

1. Aliquot 100 μL into 1.5 mL tubes on ice.
2. Optional: snap‑freeze on dry ice or liquid N₂ for 10 min; store at −80°C.
3. Electroporate or store for later use.