Competent-Cells
QC of Competent Cells
[edit]Add 500 ul of SOC medium to 100 ul competent cells
Incubate at 37 oC for 30 min. Spin down cells, leave about 100 ul liquid
Resuspend by pipetting, aliquot 50 ul to LB/Amp and LB/Kan plate.
Incubate in 37 oC o/n.
If there is any colony growing, discard this lot of competent cells.
CaCl2 Competent Cells---Do not use for cloning, maybe Ok for subcloning
[edit]1. Optional: From library efficient Top10 or STBL3, steak out a new plate and place at 37oC overnight.
2. Inoculate two to three colonies into 5 ml LBY and grow overnight at room temperature (23 to 25oC).
4. Add cells to 500 ml LBY.
5. Grow the cells at room temperature until they reach an OD600 of 0.6. The bacteria will double every 2 to 2.5 hours at 37 oC.
6. Prepare clean centrifuge tubes:
if not autoclavable:
Wash centrifuge tubes with 10% bleach, rinse with water and Millipore water.
Spray with 75% ethanol and dry in biosafty hood with fan on.
7. Transfer cells to autoclaved centrifuge tubes (450ml), and place on ice for 10 minutes.
8. Centrifuge at 3000 rpm for 5 minutes at 4oC using a pre-chilled rotor.
9. Resuspend cells gently in 30 ml of ice cold 0.1 M CaCl2, transfer to sterize/high performance 50 ml centrifuge tubes
10. Keep the cells on ice for 10 minutes.
11. Centrifuge cells at 3000 rpm for 1 min at 4oC.
12. Resuspend cells gently in 20 to 60 ml of ice cold CaGly Solution.
13. aliquote 100 ul to chilled autoclaved 100 ul tubes.
14. Freeze cells on dry ice and store at - 80 oC.
CaGly Solution.
0.1 M CaCl2
15% glycerol
Inoue Solution (10 mM PIPES, 55 mM MnCl2, 15 mM CaCl2, 250 mM KCl.):
PIPES 3 g/l,
CaCl2.2H2O 2.2 g/l,
KCl 18.6 g/l,
MnCl2.4H2O 10.9 g/l
Mix all the components except for MnCl2, adjust pH to 6.7~7.0 with KOH.
Then, dissolve MnCl2 , sterilize the solution by filtration through a prerinsed 0.45 µm filter unit and stored at 4ºC.
0.1 M CaCl2.
TSS Method
[edit]TSS Buffer
5g PEG 8000
1.5 mL 1M MgCl2 (or 0.30g MgCl2*6H20)
2.5 mL DMSO
Add LB to 50 mL
Filter sterilize (0.22 μm filter)
Grow a 5 mL seed culture of cells in LB medium to saturation.
Dilute this culture back into 25–50 mL fresh LB in a 200 mL conical flask (by at least 1/100.by at least 1/100.)
Grow the diluted culture to an OD600 = 0.2–0.5.
Put eppendorf tubes on ice now so that they are cold when cells are aliquotted into them later.
Split the culture into two 50 mL falcon tubes and incubate on ice for 10 min.
All subsequent steps should be carried out at 4°C and the cells should be kept on ice wherever possible
Centrifuge for 10 min at 3000 rpm and 4°C.
Remove supernatant. The cell pellets should be sufficiently solid that the supernatant can be just poured off.
Pipette out any remaining media.
Resuspend cells in chilled TSS buffer. The volume of TSS to use is 10% of the culture volume.
Vortex gently to fully resuspend the culture, keep an eye out for small cell aggregates even after the pellet is completely off the wall.
Higher concentrations of cells (2–3×) are found to enhance efficiency, requiring cell resuspension in 3–5% culture volume of TSS instead of 10% (Dueber & Bennett Lab).
Add 100 µL aliquots to chilled eppendorfs and store at -80°C.
RbCl-MnCl2 Method, 108 cfu/µg
[edit]Buffer A (prepare 50 ml per 500 ml culture)
Final concentrations:
100 mM RbCl2 (12 g/L)
50 mM MnCl2·4H2O (9.89 g/L)
30 mM potassium acetate (2.94 g/L)
10 mM CaCl2·2H2O (1.47 g/L)
15% (v/v) glycerol (150 ml/L)
Adjust pH to 5.8 with dilute acetic acid and filter sterilize
Note: Store at 4 °C, protect from light.
Buffer B (prepare 25 ml per 500 ml culture)
Final concentrations:
10 mM MOPS (2.09 g/L)
10 mM RbCl2 (1.2 g/L)
75 mM CaCl2·2H2O (11 g/L)
15% (v/v) glycerol (150 ml/L)
Adjust pH to 6.5 with dilute NaOH and filter sterilize
Recipe 2
Buffer A
KOAc 30 mM 2.94g
RbCl2 100 mM 12.10g
CaCl2 10 mM 1.47g,
MnCl2 50 mM 9.90g
Hexamine cobalt Cl 3 mM 0.80g
Glycerol 15% 150ml
pH 5.8 (0.2M acetic acid)
Sterilise by filtration
Buffer B
Per 200 ml
MOPS (or PIPES) 10 mM 419mg
CaCl2 75 mM 2.205g
RbCl2 10 mM 2.418g
Glycerol 15% 30ml
pH 6.5 (KOH)
Sterilise by filtration
Note: Store at 4 °C and protect from light.
- Inoculate 500 ml LB broth in a 1 L flask with 500 µl overnight E. coli culture and grow at 37 °C (200 RPM) until OD600 = 0.5 (about 4 h).
- Chill cells on ice for 10 min.
- Centrifuge E. coli cells at 2,000 x g for 10 min at 4 °C.
- Remove supernatant and resuspend the pellet in 50 ml ice-cold buffer A. Keep the cells on ice for the entire time and shake manually in circular motions on ice. It will take 20-30 min to resuspend the cells
Note: Do not pipette the cells up and down, but you may break the pellet with a pipette for faster resuspension. - Centrifuge cells at 2,000 x g for 10 min at 4 °C.
- Remove supernatant and resuspend the pellet in 25 ml ice-cold buffer B by shaking. Keep the cells on ice for the entire time. It will take some time (20-30 min) to resuspend the cells
CTAB Method for electroporation
[edit]https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4287956/
Grow E. coli (BL21DE3, XL1blue, and DH5-α), Bifidobacterium sp., and K. lactis in their respective media to (OD600 0.4–0.6) an exponential phase.
Spin down cells at 6000 ×g for 10 min at 4 °C
Resuspended at one tenth of their original volume in ice-cold 1 mM HEPES buffer.
Wash again and spin
Resuspend cells in 1 mM HEPES buffer containing 1 μg/ml (1mg/1000 ml) CTAB.
1 mg/1000 ml CTAB: dilute from 100X (100 μg/ml, 50 mg CTAB-->500 ml H2O) stock, add 5 ul of 100 μg/ml stock to 500 ml autoclaved H2O
Incubate for 15 min on ice.
Wash cells with water twice.
Resuspend cells in cold 10% glycerol solution.
Aliquot 100 μl cells
Store at -80 oC
Incubated at 4 °C for 5 min. The transformation mix was transferred to electroporation cuvettes (Bio-Rad, Hercules, and CA), and cells were electroporated in a Bio-Rad Gene Pulsar according to the manufacturer's directions for transformation of bacteria, and yeasts. The following fixed parameters of the Bio-Rad Gene Pulsar were followed, for electroporation of E. coli 0.2 cm cuvette, resistance 25 μF, and 200 V, bifidobacteria 0.2 cm cuvette, 25 μF resistance and 200 V, and yeast 0.2 cm cuvette, 25 μF resistance and 200 V. Subsequently, 0.9 ml of fresh medium was added, and the cells were grown at 37 °C. E. coli cells with shaking at 200 rpm for 1 h, Bifidobacterium sp., and K. lactis, without shaking were incubated.
Inoue Method
[edit]LB (Luria-Bertani) Medium
Per Liter:To 950 ml of deionized H2O , add Tryptone 10 g, Yeast Extract 5 g, NaCl 10 g. Shake until the solutes have dissolved. Adjust the pH to 7.0 with 5 N NaOH (~0.2 ml). Adjust the volume of the solution to 1 liter with deionized H2O . Sterilize by autoclaving for 20 minutes at 15 psi (1.05 kg/cm2) on liquid cycle.
SOB Medium
Per Liter:To 950 ml of deionized H2O , add: Tryptone 20 g Yeast Extract 5 g NaCl 0.5 g Shake until the solutes have dissolved. Add 10 ml of a 250 mM solution of KCl. (This solution is made by dissolving 1.86 g of KCl in 100 ml of deionized H2O .) Adust the pH of the medium to 7.0 with 5 N NaOH (~0.2 ml). Adjust the volume of the solution to 1 liter with deionized H2O . Sterilize by autoclaving for 20 minutes at 15 psi (1.05 kg/cm2) on liquid cycle. Just before use, add 5 ml of a sterile solution of 2 M MgCl2. (This solution is made by dissolving 19 g of MgCl2 in 90 ml of deionized H2O . Adjust the volume of the solution to 100 ml with deionized H2O and sterilize by autoclaving for 20 minutes at 15 psi (1.05 kg/cm2) on liquid cycle.)
SOB agar plates containing 20 mM MgSO4 and the appropriate antibiotic
Standard SOB contains 10 mM MgSO4.
SOB medium, for growth of culture to be transformed
Prepare three 1-liter flasks of 250 ml each and equilibrate the medium to 18-20ºC before inoculation.
SOC medium
Approximately 1 ml of this medium is needed for each transformation reaction. SOC medium is identical to SOB medium except it contains 20 mM glucose. After the SOB medium has been autoclaved, allow it to cool to 60ºC or less. Add 20 ml of a sterile 1 M solution of glucose. (This solution is made by dissolving 18 g of glucose in 90 ml of deionized H2O . After the sugar has dissolved, adjust the volume of the solution to 100 ml with deionized H2O and sterilized by passing it through a 0.22-mm filter.)
METHOD
[edit]IMPORTANT All steps in this protocol should be carried out aseptically.
Preparation of Cells
1. Prepare Inoue transformation buffer (chilled to 0ºC before use). Organic contaminants in the H2O used to prepare transformation buffers can reduce the efficiency of transformation of competent bacteria. H2O obtained directly from a well-serviced Milli-Q filtration system (Millipore) usually gives good results. If problems should arise, treat the deionized H2O with activated charcoal before use.
a. Prepare 0.5 M PIPES (pH 6.7) (piperazine-1,2-bis[2-ethanesulfonic acid]) by dissolving 15.1 g of PIPES in 80 ml of pure H2O (Milli-Q, or equivalent). Adjust the pH of the solution to 6.7 with 5 M KOH, and then add pure H2O to bring the final volume to 100 ml. Sterilize the solution by filtration through a disposable prerinsed Nalgene filter (0.45-mm pore size). Divide into aliquots and store frozen at -20ºC.
b. Prepare Inoue transformation buffer by dissolving all of the solutes listed below in 800 ml of pure H2O and then add 20 ml of 0.5 M PIPES (pH 6.7). Adjust the volume of the Inoue transformation buffer to 1 liter with pure H2O .
c. Sterilize Inoue transformation buffer by filtration through a prerinsed 0.45-mm Nalgene filter. Divide into aliquots and store at -20ºC.
2. Pick a single bacterial colony (2-3 mm in diameter) from a plate that has been incubated for 16-20 hours at 37ºC. Transfer the colony into 25 ml of LB broth or SOB medium in a 250-ml flask. Incubate the culture for 6-8 hours at 37ºC with vigorous shaking (250-300 rpm).
3. At about 6 o'clock in the evening, use this starter culture to inoculate three 1-liter flasks, each containing 250 ml of SOB. The first flask receives 10 ml of starter culture, the second receives 4 ml, and the third receives 2 ml. Incubate all three flasksovernight at 18-22ºC with moderate shaking.
4. The following morning, read the OD600 of all three cultures. Continue to monitor the OD every 45 minutes.
5. When the OD600 of one of the cultures reaches 0.55, transfer the culture vessel to an ice-water bath for 10 minutes. Discard the two other cultures.
The ambient temperature of most laboratories rises during the day and falls during the night. The number of degrees and the timing of the drop from peak to trough varies depending on the time of year, the number of people working in the laboratory at night, and so on.
Because of this variability, it is difficult to predict the rate at which cultures will grow on any given night. Using three different inocula increases the chances that one of the cultures will be at the correct density after an overnight incubation
6. Harvest the cells by centrifugation at 2500g (3900 rpm in a Sorvall GSA rotor) for 10 minutes at 4ºC.
7. Pour off the medium and store the open centrifuge bottle on a stack of paper towels for 2 minutes. Use a vacuum aspirator to remove any drops of remaining medium adhering to walls of the centrifuge bottle or trapped in its neck.
8. Resuspend the cells gently in 80 ml of ice-cold Inoue transformation buffer.The cells are best suspended by swirling rather than pipetting or vortexing.
9. Harvest the cells by centrifugation at 2500g (3900 rpm in a Sorvall GSA rotor) for 10 minutes at 4ºC.
10. Pour off the medium and store the open centrifuge tube on a stack of paper towels for 2 minutes. Use a vacuum aspirator to remove any drops of remaining medium adhering to the walls of the centrifuge tube or trapped in its neck.
Freezing of Competent Cells
11. Resuspend the cells gently in 20 ml of ice-cold Inoue transformation buffer.
12. Add 1.5 ml of DMSO. alternatively, add βmercaptoethanol to 20 mM final concentration (add 8 µL of 100% βmercaptoethanol to 20 ml). Mix the bacterial suspension by swirling and then store it in ice for 10 minutes.
for competent cells without βmercaptoethanol, add 2 µl of 0.5 M βmercaptoethanol to 50 µL competent cells (20 mΜ final) prior to transformation.
for competent cells without DMSO & β mercaptoethanol, add 8 µl of 0.5 M βmercaptoethanol and 15 µl DMSO to 200 µL competent cells (20 mΜ final) prior to transformation.
13. Working quickly, dispense aliquots of the suspensions into chilled, sterile microfuge tubes. Immediately snap-freeze the competent cells by immersing the tightly closed tubes in a bath of liquid nitrogen. Store the tubes at -70ºC until needed.Freezing in liquid nitrogen enhances transformation efficiency by ~5-fold. For most cloning purposes, 50-ml aliquots of the competent-cell suspension will be more than adequate. However, when large numbers of transformed colonies are required (e.g., when constructing cDNA libraries), larger aliquots may be necessary.
14. When needed, remove a tube of competent cells from the -70ºC freezer. Thaw the cells by holding the tube in the palm of the hand. Just as the cells thaw, transfer the tube to an ice bath. Store the cells on ice for 10 minutes.
15. Use a chilled, sterile pipette tip to transfer the competent cells to chilled, sterile 17 x 100-mm polypropylene tubes. Store the cells on ice. Glass tubes should not be used since they lower the efficiency of transformation by ~10-fold
For competent cells without DMSO, add 7.5 ul DMSO to 100 ul competent cells after thawing on ice
Transformation
Include all of the appropriate positive and negative controls
16. Add the transforming DNA (up to 25 ng per 50 ml of competent cells) in a volume not exceeding 5% of that of the competent cells. Swirl the tubes gently several times to mix their contents. Set up at least two control tubes for each transformation experiment, including a tube of competent bacteria that receives a known amount of a standard preparation of superhelical plasmid DNA and a tube of cells that receives no plasmid DNA at all. Store the tubes on ice for 30 minutes.
17. Transfer the tubes to a rack placed in a preheated 42ºC circulating water bath. Store the tubes in the rack for exactly 90 seconds. Do not shake the tubes.Heat shock is a crucial step. It is very important that the cells be raised to exactly the right temperature at the correct rate. The incubation times and temperatures given here have been worked out using Falcon 2059 tubes. Other types of tubes will not necessarily yield equivalent results.
18. Rapidly transfer the tubes to an ice bath. Allow the cells to cool for 1-2 minutes.
19. Add 800 ml of SOC medium to each tube. Warm the cultures to 37ºC in a water bath, and then transfer the tubes to a shaking incubator set at 37ºC. Incubate the cultures for 45 minutes to allow the bacteria to recover and to express the antibiotic resistance marker encoded by the plasmid.To maximize the efficiency of transformation, gently agitate (<225 cycles/minute) the cells during the recovery period.
20. Transfer the appropriate volume (up to 200 ml per 90-mm plate) of transformed competent cells onto agar SOB medium containing 20 mM MgSO4 and the appropriate antibiotic.When selecting for resistance to tetracycline, the entire transformation mixture may be spread on a single plate (or plated in top agar). In this case, collect the bacteria by centrifuging for 20 seconds at room temperature in a microfuge, and then gently resuspend the cell pellet in 100 ml of SOC medium by tapping the sides of the tube.IMPORTANT Sterilize a bent glass rod by dipping it into ethanol and then in the flame of a Bunsen burner. When the rod has cooled to room temperature, spread the transformed cells gently over the surface of the agar plate. When selecting for resistance to ampicillin, transformed cells should be plated at low density (<104 colonies per 90-mm plate), and the plates should not be incubated for more than 20 hours at 37ºC. The enzyme b-lactamase is secreted into the medium from ampicillin-resistant transformants and can rapidly inactivate the antibiotic in regions surrounding the colonies. Thus, plating cells at high density or incubating them for long periods of time results in the appearance of ampicillin-sensitive satellite colonies. This problem is ameliorated, but not completely eliminated, by using carbenicillin rather than ampicillin in selective media and increasing the concentration of antibiotic from 60 mg/ml to 100 mg/ml. The number of ampicillin-resistant colonies does not increase in linear proportion to the number of cells applied to the plate, perhaps because of growth-inhibiting substances released from the cells killed by the antibiotic
21. Store the plates at room temperature until the liquid has been absorbed.
22. Invert the plates and incubate them at 37ºC. Transformed colonies should appear in 12-16 hours.
Prepare Inoue transformation buffer (chilled to 0°C before use).
[edit]| Reagent |
Amount per Liter |
Final Concentration |
| MnCl2 • 4H2O |
10.88 g |
55 mM |
| CaCl2 • 2H2O |
2.20 g |
15 mM |
| KCl |
18.65 g |
250 mM |
| PIPES (0.5 M, pH 6.7) |
20 ml |
10 mM |
| H2O to 1 liter |
c. Sterilize Inoue transformation buffer by filtration through a prerinsed 0.45-μm Nalgene filter. Divide into aliquots and store at –20°C.
REFERENCES
Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166: 557-580.
Hanahan, D. 1985. Techniques for transformation of E. coli. In DNA cloning: A Practical Approach (ed. D.M. Glover), vol. 1 pp. 109-135. IRL Press, Oxford, United Kingdom.
Inoue H., Nojima H., and Okayama H. 1990. High efficiency transformation of Escherichia coli with plasmids. Gene 96: 23-28. NEB
Electrocompetent Cells
[edit]Sterile 10% glycerol (can be autoclaved) is needed for the washes. The volume of 10% glycerol needed is 2X the culture volume (for example, a 500 ml culture requires 1L of 10% glycerol).
Procedure (for 2, 250 ml cultures)
[edit]
Inoculate 1 colony from a fresh plate of the strain to be made electrocompetent into 10 ml of SOB in a 125 ml flask and incubate for 16-18 hours at 37oC and 250 rpm.
Have ready 2, 1 L flasks containing 250 ml each of SOB pre-warmed to 37°C. Add two drops of the overnight culture to each of the flasks.
Shake at 37°C and 250 rpm until the cultures reach an OD600 of 0.5-0.7. Be sure to turn on centrifuge and cool rotor to 4°C well in advance of harvesting cells. Be sure to place 1 L of 10% glycerol on ice well in advance of harvesting cells
Place cultures on ice for 15 minutes. From this point on the cultures must be kept ice cold. Pour each 250 ml culture into chilled 500 ml (or 1000 ml) centrifuge bottles.
Centrifuge at 5000 rpm for 10 min. Pour off the supernatant and aspirate any residual broth.
Add 250 ml of glycerol to each of the centrifuge bottles and completely suspend the cells by pipetting up and down.
Centrifuge at 5000 rpm for 10 min. Pour off the supernatant, it is not necessary to aspirate. Completely suspend the cells in 250 ml glycerol and re-centrifuge.
Pour off the supernatant and suspend the cells in the residual glycerol by pipetting up and down.
At this point you can electroporate or freeze the cells away. To freeze, add 100 microliters of the culture to microcentrifuge tubes on ice. Once you have used all of the culture, transfer the tubes to dry ice for 10 minutes. Once the cultures are frozen, transfer them to a -80°C freezer. The cultures should be good for >6 months.
Electroporation Protocol
[edit]Ligation at 16 oC o/n,
Add 1 µl of 1 µg/µl yeast tRNA, 50 µl 100% cold ethanol, put into −20°C freezer for 15 min
Spin 10 min
Wash with 100 µl of 70% ethanol
Air-dry
Resuspend in 1 µl of ultrapure water.
Transform with 20 µl electrocompetent cells
The electroporation protocol will vary depending on the strain so this protocol may need to be optimized. For control electroporation dilute pUC19 to 10 pg/µl with Milli-Q water.
Turn on electroporator and set to 1.7-2.5 kv (optimize for strain), 200 ohms and 25 µF.
Place recovery SOC in 37°C water bath.
Pre-warm LB-antibiotic plates at 37°C.
Thaw cells on ice for 10 min or use freshly made cells.
Place appropriate number of microcentrifuge tubes and 1 mm-electroporation cuvettes on ice.
Flick the tube containing cells a few times to mix and add 25 µl to the microcentrifuge tubes.
Add 1 µl of a 10 pg/µl DNA solution (in DI water) to the cells in the microcentrifuge tube.
Transfer the DNA-cell mixture to the cold cuvette, tap on countertop 2X, wipe water from exterior of cuvette and place in the electroporation module and press pulse (don’t hold the button down).
Immediately add 975 µl of 37°C SOC, mix by pipetting up and down once and transfer to a 15 ml-falcon tube.
Rotate in the 37°C incubator for 1 h.
Make appropriate dilutions. When using 10 pg of DNA, make two dilutions:
Dilute 10 µl cells into 990 µl SOC and plate 100 µl. (1000-fold dilution)
Dilute 100 µl cells into 900 µl SOC and plate 100 µl. (100-fold dilution)
Incubate overnight at 37°C.
Calculation:
If the culture was diluted 1000-fold when plated, the total cfu per ml is 1000 times the number of colonies counted. The cfu is divided by the amount of pUC19 (10 pg per ml)
cfu/ µg = (colonies counted*1000) / (0.00001 µg pUC19)
Procedure
•Inoculate 250 mL SOB media with 10 - 12 colonies from a freshly streaked plate.
•Grow to an OD600 of 0.94 at 18°C (or RT). (Doubling time is 4-10 hours). (1/04: 9.7 hr for INVaF’)
•Chill on ice for about 10 minutes
- From this point on, it is critical to keep cells below 4°C**
- Work in cold room when possible**
•Pellet cells at 2500 x g for 10 minutes at 4°C.
•Resuspend cells gently in 80 mL of ice cold TB.
•Leave on ice for ~10 min.
•Pellet cells as before
•Resuspend cells gently in 10 mL ice cold TB.
•Add 10 mL ice cold TB + 14% DMSO.
•Wait 10 minutes, then aliquot into pre-cooled 1.5 mL tubes, and freeze them in liquid nitrogen.
•Store in -70 oC freezer.
Transformation
•Thaw cells on ice. (If desired, split comp cells to pre-cooled tubes. Can use 12.5 µL/transformation.)
•(Optional for ~2-3X efficiency increase: Add 4 µL of 0.5 Μ (or 0.14 µL of 100%) β-mercaptoethanol to cells per 100 µL competent cells. (20 mΜ final)
•Add DNA (I add <3 µL per 100 µL comp cells)
Incubate in ice for ~30 minutes. (Longer incubations seem to help for plasmids >10 kb.)
•Heat shock at 42°C for 30 seconds then place on ice for another 2 minutes.
•Add 0.9 mL pre-warmed SOC media. (LB works but might affect efficiency)
• 37°C for 45 minutes (or longer).
(Some people suggest shaking, others say it kills the cells)
•Plate
Protocol 2 Electrocompetent Cells
[edit]Day 1
1. Streak out frozen glycerol stock of bacterial cells (Top 10, DH5α, etc.) onto an LB
plate (no antibiotics). Grow plate overnight at 37°C.
Day 2
1. Autoclave:
2 L of ddH2O
100 mL of 10% v/v glycerol (molecular biology grade)
1 L LB (or your preferred media)
4 centrifuge bottles and caps
Lots of microfuge tubes
2. Chill overnight at 4°C:
ddH2O
10% glycerol
Centrifuge rotor
3. Prepare starter culture of cells
Select a single colony of E. coli from fresh LB plate and inoculate a 10 mL starter
culture of LB (or your preferred media). Grow culture at 37°C in shaker overnight.
Notes:
You can also substitute other media like SOB, 2xYT, etc for LB if preferred. All
glassware should be detergent free. Trace detergent residue reduces
competency.
Day 3
1. Inoculate 1 L of LB media with 10 mL starter culture and grow in 37°C shaker.
Measure the OD600 every hour, then every 15-20 minutes when the OD gets
above 0.2.
2. When the OD600 reaches 0.35-0.4, immediately put the cells on ice. Chill the
culture for 20-30 minutes, swirling occasionally to ensure even cooling. Place
centrifuge bottles on ice at this time.
IMPORTANT NOTES:
• It is important not to let the OD get any higher than 0.4. The OD should be
carefully monitored and checked often, especially when it gets above 0.2, as the
cells grow exponentially. It usually takes about 3 hours to reach an OD of 0.35
when using a 10 mL starter culture.
• It is also very important to keep the cells at 4°C for the remainder of the
procedure. The cells, and any bottles or solutions that they come in contact with,
must be pre-chilled to 4°C.
3. (Spin #1) Split the 1 L culture into four parts by pouring about 250 mL into ice
cold centrifuge bottles. Harvest the cells by centrifugation at 1000g (~2400 rpm
in the Beckman JA-10 rotor) for 20 minutes at 4°C.
4. Decant the supernatant and resuspend each pellet in 200 mL of ice cold ddH2O.
5. (Spin #2) Harvest the cells by centrifugation at 1000g (~2400 rpm in the
Beckman JA-10 rotor) for 20 minutes at 4°C.
6. Decant the supernatant and resuspend each pellet in 100 mL of ice cold ddH2O.
7. (Spin #3) Combine resuspensions into 2 centrifuge bottles (so each contains
about 200 mL of cell suspension). Harvest the cells by centrifugation at 1000g
(~2400 rpm in the Beckman JA-10 rotor) for 20 minutes at 4°C. At this step,
rinse two 50 mL conical tubes with ddH2O and chill on ice.
8. Decant the supernatant and resuspend each pellet in 40 mL of ice cold 10%
glycerol. Transfer each suspension to a 50 mL conical tube.
9. Harvest the cells by centrifugation at 1000g (~2100 rpm in the Beckman GH-3.8
rotor) for 20 minutes at 4°C. Start putting 1.5 mL microfuge tubes on ice if not
already chilled.
10. Carefully aspirate the supernatant with a sterile Pasteur pipette (pellets lose
adherence in 10% glycerol). Resuspend each pellet in 1 mL of ice cold 10%
glycerol by gently swirling. The final OD600 of the resuspended cells should be ~
200-250.
11. Aliquot into sterile 1.5 mL microfuge tubes and snap freeze with liquid nitrogen.
Store frozen cell in the -80°C freezer.
SOC Medium
Add 20 ml of sterile 1 M glucose per liter of SOB medium immediately before use.
Or make frozen aliquots and store at -20 ºC.
SOB Medium
1. Measure ~900ml of distilled H2O
2. Add 20g Bacto Tryptone
3. Add 5g Bacto Yeast Extract
4. Add 2ml of 5M NaCl
5. Add 2.5ml of 1M KCl
6. Add 10ml of 1M MgCl2
7. Add 10ml of 1M MgSO4
8. Adjust pH to 7.0 with 10N NaOH and adjust volume to 1 L with distilled H2O.
9. Autoclave to sterilize on LIQUID cycle 15 minutes.
2xYT Medium
1. Measure ~900ml of distilled H2O.
2. Add 16g Bacto Tryptone.
3. Add 10g Bacto Yeast Extract.
4. Add 5g NaCl.
5. Adjust pH to 7.0 with 5N NaOH.
6. Adjust to 1L with distilled H2O.
7. Sterilize by autoclaving on LIQUID cycle.
LB (Luria-Bertani) Broth Recipe
10 grams tryptone
5 grams of yeast extract
10 grams of NaCl
(NOTE: A premixed powder is also available from Fisher cat# BP1426)
1. Dissolve in 1 L of water.
2. Portion into flasks and cover will aluminum foil. Fill the flask to half its volume.
3. Autoclave 20-30 minutes on LIQUID cycle.
Protocol 3 Making Electrocompetent E. coli Cells
[edit]
small batch for 2-3 transformations.
Grow an overnight culture of each strain in LB medium.
Prepare 10 ml of fresh LB medium in a 50 ml flask for each strain.
Inoculate with 100 μl of the overnight, stationary-phase culture.
In general, inoculate to OD600 of ~0.05.
Grow the cells for approximately 2-3 hours, until they reach mid-exponential phase.
In general, this is an OD600 of ~0.6.
Transfer the cells to 15 ml Falcon conical tubes.
Pellet the cells by centrifugation for 5 minutes at 6,000 RPM. Remove promptly and pour off supernatant.
Wash by adding 10 ml of chilled 10% glycerol to each tube, then vortexing vigorously to resuspend the pellet. Centrifuge for 3.5 minutes. Remove promptly and pour off supernatant. Repeat for at least four wash cycles in 10% glycerol.
Resuspend in approximately 100 μl of 10% glycerol to make a 100x concentration of the initial culture.
Divide into 30-50 μl aliquots in 0.5 or 1.7 ml tubes. Freeze or proceed directly to electroporation.
Notes
Keeping the cells cold during all processing steps is recommended. Ideally, chill the subculture in an ice water bath for >10 min before centrifuging, use a refrigerated centrifuge or a centrifuge in a cold room, and aliquot the final cell slurry into pre-chilled tubes for storage at -80C. These precautions are not strictly necessary if using the cells for routine cloning.
Scale up the culture volumes if more electrocompetent cells are needed. The volume of 10% glycerol used for each wash should be at least 20× the volume of the cell pellet.
Electrocompetent E. coli Cells, large batch
Grow an overnight culture of each strain in LB medium.
Prepare 100 ml of fresh LB medium in a 500 ml flask for each strain.
Inoculate with overnight, stationary-phase culture to an OD of ~0.05.
Grow the cells for approximately 2-3 hours, until they reach mid-exponential phase.
In general, this is an OD600 of 0.4-0.6.
~30 min before you plan to prepare your cells, set the centrifuge to 4C.
Transfer the cells to 2x 50 ml Falcon conical tubes.
Pellet the cells by centrifugation for 5 minutes at 6,000 RPM. Remove promptly and pour off supernatant.
Wash by adding 40 ml of chilled 10% glycerol to each tube, then vortexing vigorously to resuspend the pellet. Centrifuge for 3.5 minutes. Remove promptly and pour off supernatant. Repeat for at least four wash cycles in 10% glycerol.
Resuspend each pellet in approximately 500 μl of 10% glycerol to make a 100x concentration of the initial culture.
Divide into 30-50 µl aliquots in 0.5 or 1.7 ml tubes. Freeze promptly at -80C.
Transforming E. coli Cells by Electroporation
This procedure uses the TOP10 Electrocomp™ E. coli cells (but is identical in any other standard electromp cell type).
Thaw the electrocompetent cells on ice.
To the electrocompetent cells, add 1-3 μl of DNA (<100 ng of DNA).
Mix by gently flicking the tube containing the electrocompetent cell + DNA mixture. Let the mixture sit on ice for 1-10 minutes.
Pipette the mixture into a chilled cuvette, making sure that the mixture is at the bottom of the cuvette by gently tapping the cuvette on a flat surface.
Be sure to wipe any condensation off the sides of the cuvette before electroporation.*
Place the cuvette in the pulser and press the "Pulse" button.
For the TOP10 Electrocomp™ E. coli cells, the Ec l setting is fine.
After electroporation, add 500-1000 μl of SOC/LB to the cuvette to recover the cells.
Transfer the mixture to a 1.5 mL microcentrifuge tube.
Incubate for ~30-60 minutes at 37°C or other appropriate temperaturein a shaking incubator.
Be sure to place the tube on its side so the transformed cells will grow properly.
Plate the cells (~ 50 μl) on an LB plate containing the appropriate antibiotic.
Incubate overnight at 37°C or other appropriate temperature.