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Competent Cell Preparation

Choice of protocol parameters

• Choose the number of transformation aliquots to prepare n, and choose the volume V of each, typically 50 µL for 1 mm cuvettes, though ≤90 µL can be loaded in the gap of 1 mm cuvettes and ≤400 µL in 2 mm cuvettes (including added DNA volume).
• Choose the cell resuspension concentration, defaulting to OD 50, which requires 125-fold concentration of the log-phase ≈OD 0.4 culture, thus requiring growing a 125nV mL culture. 
• Choose a broth and corresponding growth temp based on supporting protocols and required efficiency:
  LB-Lennox (lysogeny broth, 5 g/L NaCl) or 2×YT can be used for routine high-efficiency work with small/medium-sized plasmids ⁽³⁾. LB-Miller (10 g/L salt) also works fine. Grow at 37°C. Consider using SOB(Mg^–)⁽²⁾ for more reliably high resistance.
  SOC-sucrose adds glucose and sucrose to SOB, which when used in 21°C culture was found to greatly boost efficiency with large plasmid (>50 kb) transformation.
• The default three ½-volume glycerol washes may be reduced to two, or ½-¼-¼ washes if large libraries/DNAs are not intended. Washes in HEPES instead of glycerol might be experimented with ⁽⁵⁾.
• GYT medium for final resuspension might be experimented with for higher efficiency ⁽⁴⁾⁽⁶⁾.

For n transformations of V volume with X-fold concentration of the culture:

Materials

1. Culture broth of choice, nVX mL: 

   LB-Lennox (Lysogeny Broth) or 2×YT or
   SOB(Mg^–)(use at 37°) or SOC-Sucrose (use at 21°):

   Tryptone	20 g/L	–	Dissolve with stir bar. Check pH.
   Yeast extract	5 g/L	–
   NaCl	0.5 g/L	8.56 mM
   KCl	0.746 g/L	2.5 mM
   Milli-Q water	Reserve volume for Mg and sugar solns
   NaOH, 1 M	→ pH 7.0
   Autoclave 15 min ≤ 1 L, 20 min 1–1.4 L. Or filter sterilize* after adding components below.
   MgCl2 sterile	10 mL/L 1 M	10 mM	Autoclaved or filtered*. Omit for Mg– version.
   MgSO4 sterile	10 mL/L 1 M	10 mM
   Glucose sterile	20 mL/L 1 M 7.2 mL/L 50% 18 mL/L 20%	20 mM 3.6 g/L	For SOC-sucrose only. Autoclaved or filtered*.
   Sucrose sterile	80 mL/L 2.5 M 137 mL/L 50%	200 mM	For SOC-sucrose only. Autoclaved or filtered*.
   Store at 4° for best long-term preservation of nutrients. Aliquots of SOC can be frozen to inhibit contaminant growth. * Detergent-free membranes (PES, NYL, CN) are preferred for filter-sterilization.

   
   

2. Recovery medium: SOC (best), LB or 2×YT (ok, good with added glucose and Mg)
3. 10% glycerol, ≥1.5 · nVX mL.
   Culture volume × 1½, since three ½-volume washes are prescribed here, though this may be reduced if using one less wash or reducing the sizes of the last two washes.
4. If you want to try alternative final resuspension/electroporation medium (1 mM HEPES pH 7.0 or GYT medium), prepare nV mL of it. GYT is 10% glycerol, 1.25 g/L yeast extract, 2.5 g/L tryptone, filter-sterilized and stored 4°C.
5. Culture tubes for precultures.
6. Culture flasks large enough to hold growth medium volume.
7. Shaking incubator space for the culture flasks, refrigerated if using low-temp growth protocol.
   • Only a refrigerated shaker can sustain 18–23°C temperature.
8. Ice and ice bucket/tray large enough to swirl flasks in after growth.
9. Chilled centrifuge bottles/lids or tubes appropriate for holding culture volumes and balancing.
   • Bottles must be able to collectively hold culture(s) without any exceeding 80% capacity. Filling bottles higher will result in leakage into rotor.
10. Chilled sterile water to balance multiple centrifuge bottles, if needed.
11. 4°C centrifuge and rotor compatible with centrifuge bottles.
12. Labeled cryoboxes for comp cell aliquots, prechilled at -80°C.
13. (opt.) Liquid nitrogen, dewar, and slotted ladle, if flash freezing.
14. Cold block or ice for holding aliquot tubes; ice for holding centrifuge bottles and resuspension tubes.
    Alternatively: do final cell resuspension and aliquotting in cold room (4°), using mobile lab bench detailed below. Cold room prep is practically essential for larger scale comp cell preps, it can otherwise be very cumbersome to keep the needed number of aliquot tubes ready on ice or cold blocks. Tubes are difficult to store and manipulate on ice while keeping cold, especially 0.2 mL tubes, and labs only have a few cold blocks if at all.
15. Materials at the work bench or on a mobile lab bench cart for the cold room:
    1. n tubes (e.g. 0.2 mL tubes) for cell aliquots, labeled/marked and arranged in clean tube racks. Blade, if planning to cut apart tube strips.
    2. Extra bag of tubes, for aliquotting any excess comp cell volume, if desired.
    3. Pipettes/tips appropriate for resuspending and transferring cells 
    4. A 5 or 50 mL tube per strain to hold resuspension.
    5.  (opt.) Electronic repeater pipette and a 5 mL combitip per strain and some extras. Reservoirs, if opting to use a multichannel pipette.
    6. Micropipette and tips appropriate for aliquot volumes ≥v. (Useful to use up residual volume after using electronic repeater pipette.)
    7. Ethanol spray
    8. Paper towels
    9. Extra gloves
    10. Trash bin/bag
    11. (opt.) If flash freezing, box/bag to collect all tubes in before flash-freezing.
        

Protocol

1. Grow preculture of strain to saturation:
   Inoculate a couple mL of chosen broth (~¹∕₄₀₀ nVX culture volume _{+antibiotics if needed}) with colonies (best) or a scrape of frozen stock of the desired strain, and incubate shaking 37°C 8–16 hr until saturated.
   • Use a fresh, trusted source of the strain. For most reliable results, inoculate a seed culture from colonies/patch from <2 week-old plates streaked from a frozen stock. Plasmid-bearing strains ought to be used from even fresher plates to minimize plasmid mutations.
   • Meanwhile, prewarm main culture broth if doing 37° growth.
2. Measure Grow culture: measure preculture OD. Inoculate main culture volume ( nvX mL) of chosen broth with ≈0.2–0.4% saturated seed culture, to a final OD₆₀₀≈0.01–0.02.
   Incubate 37°C or 18–22°C (depending on protocol), shaking 250 rpm for flasks.
   Grow culture to OD₆₀₀=0.4–0.45 (exponential phase), 3–4 hr at 37°C or 9–15 hr for low-temp. Periodically monitor OD an hour before the range begins.
   • Cloning strains (rec^–) are slower; wilder strains are faster.
   • ≈12–15 hr required for 18° culturing, less for higher temperatures.
3. Prepare the mobile lab bench cart for the cold room as described. Prechill both the mobile lab bench cart (with its contents) and the centrifuge to 4°C ≥45 min before cell harvest (next step).
4. When Growth arrest: when the culture reaches OD₆₀₀≈0.4, quickly move the flask to an ice-water bath and swirl for 10–15 min for large volumes, or until the culture is ice-cold.
   • This pre-chilling step can probably be omitted except for the higher efficiencies.
5. Harvest cells: decant into prechilled centrifuge bottles and balance them. Use chilled sterile water if necessary.
   Centrifuge 1000×g, 10–20 min (depending on volume) in a prechilled 4°C centrifuge.
   • 800×g 20 min or 2500×g 5–10 min, depending on volume.
6. Check for a pellet. Gently decant Decant medium away from pellet gently, shaking the bottle to drain, but taking care not to lose the pellet. Absorb the last of the medium on the lip with clinging to the bottle walls by striking the inverted bottle down on a paper towel and/or aspirating drops away.
   Quickly return bottles to ice.
7. Gently resuspend cells in ½ the original culture volume (½nVX mL) of chilled 10% glycerol, by swirling.
   Centrifuge as in step 5.
8. Wash cells again (step 6–7).
9. Wash cells again (step 6–7).
   • You might opt to eliminate the third wash or reduce the last two wash volumes to ¼ the culture volume (¼nVX mL) as in some protocols. And/or experiment doing all washes in chilled 1 mM HEPES pH 7.0 as in ⁽⁵⁾.
10. Gently resuspend Final resuspension: resuspend gently in desired final volume (nv mL) chilled 10% glycerol by swirling and striking, while keeping cold.
    
    • Flat-bottom centrifuge bottles allow easier gentle resuspension of pellets.
    • If even higher efficiency is desired, you might experiment with using GYT medium here instead ⁽⁴⁾⁽⁶⁾. Or 1 mM HEPES pH 7.0 as in (5), but aliquot enough for immediate electroporation at purported higher efficiency, and spike in 50% glycerol to a final 10% for frozen storage of remaining cells.
    
    Perform remaining steps with pre-chilled materials in the cold room for large scale preps and maximum efficiency.
    Decant or pipette cells into 5 or 50 mL conical tubes on ice for easier access with pipette for aliquotting. Finish gently resuspending any visible remaining cell clumps using P1000 tip if necessary.
11. Aliquot into tubes in the cold room (for top efficiency) or on cold block or ice. You can use a repeater pipette or multichannel pipette from a chilled reservoir.
    Cap the tubes while they are racked, and slice apart if using tube strips. Minimize touching the tubes to keep them cool. Check that all caps are fully in the tube.
    Dislodge tubes from racks into a bag/box without touching the bottom (your hands are warm). This is easiest by pushing them out from the bottom using a spare rack or tip box.
12. Flash freeze (opt.) Flash freeze in liquid nitrogen for supposedly reportedly higher efficiency.
    • Flash freezing improves competenceis reported to retain higher competence of cells in the literature, but simply freezing is not estimated to noticeably greatly reduce competence, maybe within twofold twofold ^[SPB].
    • Residual ethanol from For a dry-ice-ethanol bath, residual ethanol fails to dry from tubes/boxes at -80°C. Freeze tubes in a bag if using this method, and pour tubes into box.
13. Quickly Store: quickly move tubes to prechilled, labeled -80°C freezer boxes, either directly from 4°C, or if flash freezing, directly ladled out of liquid nitrogen dewar or dry-ice/ethanol bag (so as not to heat tubes).
    If not freezing, proceed to transformation right away.

Detergent Residue

See section in Chemical/Heat-Shock transformation (CCMB80 method)OM_Eporator_4309_900_010-05_052016_en.fm (eppendorf.com)

Transformation

Summary

1. Thaw comp cell aliquots (50–90 µL) slowly on ice or above cold block for a few min.
   You may dilute an aliquot 2–4× with cold 10% glycerol to get more reactions out of it.
   Prechill electroporation cuvettes and DNA on ice.
   Prewarm recovery medium to 37° (opt.)
   Label recovery tubes or block seal.
2. Add pure DNA to cells and mix twice by pipetting.
3. Incubate cold on 1°C block / ice for 1–5 min (opt)
4. Load cell-DNA suspension into chilled cuvette.
5. Electroporate: tap cuvette on table, wipe electrodes, and immediately pulse at 1.7 kV for 1 mm cuvette.
6. Recovery: within a few seconds, resuspend cells in 0.5–1 mL SOC and move to recovery vessel.
   Incubate at growth temp, 1 hr shaking, 2–3 hr for nonselective chromosomal edits.
7. Plate on selective agar and incubate appropriately.

Detailed

1. Thaw comp cell aliquots (50–90 µL) slowly on ice or above cold block for a few min.
   • You may dilute an aliquot 2–4× with cold 10% glycerol to get more reactions out of it.
   • These may be stored in 0.2 mL "PCR" tubes to minimize space.
   Prechill electroporation cuvettes and DNA on ice. 1 mm cuvettes can hold 90 µL.
   Prewarm  recovery medium to 37–42° (optional, enhances efficiency)
   Label recovery tubes or 24/96-well plate seal.
   Pre-aliquot recovery medium in recovery tubes/plate and warm in a heated block or water/bead bath (opt.)
2. Add pure DNA to cell aliquot and mix twice by pipetting.
   • Purified DNA in Tris buffer works as well as DNA in water. 10–50 pg supercoiled plasmid ⁽⁴⁾. Libraries, up to 1 µg ⁽³⁾. See sources for data on TE vs DNA quantity.
   • 0.5 µL of unpurified DNA assembly or digest reaction still gives high enough colony yield for complex cloning or small libraries (1E3 CFU) despite the enzymes and salts, or even high-salt NEBuffer 3.1. Column purification, however, allows all the DNA, less the ~30% purification loss, to be electroporated, and at maximal efficiency in the absence of the salts and enzymes ^(Shyam). You might alternatively dilute assemblies 2–5-fold first to reduce salt input.
   • If avoiding cotransformation events, consider reducing DNA quantity, <10 ng DNA / mL total volume ⁽¹⁾⁽⁴⁾.
   • Enzyme inactivation may greatly enhance efficiency. Ligase heat-inactivation was found to increase efficiency 260-fold ⁽⁵⁾.
3. Incubate cold on 1°C block or ice for 1–5 min (opt.)
   • Purported to raise efficiency somewhat. Skip if cells produce extracellular DNases (EndA) which may degrade DNA.
   • More than 1 min does not increase efficiency⁽¹⁾. Elsewhere, 0 ⁽⁴⁾ or 5 min ⁽³⁾ is used without explanation.
4. Load cell suspension between cuvette electrodes while on ice.
   • Avoid bubbles during pipetting, as they may cause arcing. Pipette cells into one end of the gap against the clear wall.
5. To electroporate, in quick succession before the cuvette warms up do the following:
   Tap cuvette firmly on table to level cells into the bottom of the well.
   Wipe electrodes with paper towel to remove moisture.
   Load cuvette into electroporator cuvette holder in proper orientation and reinsert.
   • Align cuvette notch with slit in cuvette holder.
   Pulse at 1.7 kV for 1 mm cuvette, 2.5 kV for 2 mm cuvette.
   • For E. coli, optimum fields are around 1.2–1.9 kV/mm.
   • If a pop is heard, arcing has occurred. Discard the cuvette and try again with less DNA or a cleaner, less salty DNA sample. If a clean, control DNA arcs, the comp cells may have too low resistance to use.
   • Eppendorf Eporator: default P1 program for 1.7 kV, P2 program for 2.5 kV
6. Recovery: within a couple seconds, resuspend cells in 0.3–1 mL recovery medium (SOC) and move to recovery vessel.
   
   • Have a pipette tip loaded or even prefilled with recovery medium.
   • A 1 min delay in adding recovery medium reduces efficiency 3-fold; 20-fold after 10 min.
   • 37–42°C recovery medium enhances efficiency. You can pre-aliquot it and keep it in a bath/block next to the electroporator.
   Incubate at growth temp (e.g. 37°C), 1–2 hr shaking, or 2–3 hr for nonselective chromosomal modifications.
7. Plate on selective agar. For colony isolation, plate only a fraction of the recovery culture and streak generously to single colonies, or streak/spread dilutions; high transformation efficiency can otherwise produce a lawn of cells. Large library plates or entire autoclave bins can be used for generating lawns of cells for libraries.
   Incubate plate appropriately.

To wash and reuse electroporation cuvettes:

• Fill cuvette with 10% bleach to brim, no more than 20 min.
  Pour out bleach and flick out remnants.
• Fill with tap water; flick out water × 5
• Fill with MilliQ water; flick out water × 5
• Dry in a 37° rack
  You may not want to reuse cuvettes in which arcing has occurred, as it might have damaged the cuvette.