Transformation of E. coli Cells Using an Adapted Calcium Chloride Procedure

This protocol describes the preparation and transformation of competent E. coli DH5α using an adaptation of the calcium chloride procedure (12).

1. Set-up

1. Equipment
   • Spectrophotometer
   • Sorval Centrifuge (or equivalent)
   • Benchtop centrifuge
   • Heat block or water bath
   • Orbital Shaker
   • Stationary Incubator
   • Gel casting tray
   • Well combs
   • Voltage source
   • Gel box
   • UV light source
   • Microwave
2. Solutions and reagents
   • Luria-Bertani (LB) broth (10 g casein enzymic hydrolysate, 5 g yeast extract and 5 g sodium chloride in 1000 mL of H₂O)
   • Super Optimal broth with Catabolite repression (SOC): (2% (w/v) tryptone, 0.5% (w/v) yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, and 20 mM glucose)
   • CaCl₂-MgCl₂ (80 mM MgCl₂, 20 mM CaCl₂) solution.
   • M CaCl₂ solution (if cells will be transformed immediately) or 0.1M CaCl₂ solution containing 10% (v/v) glycerol (if cells will be frozen for future use).
   • LB agar plates
   • LB agar selective plates (for this experiment, since the plasmid used confers ampicillin resistance, LB agar plates containing ampicillin 100 µg/mL were used)
   • E. coli DH5α strain
   • Plasmid pUC19 DNA (100 pg/ µl)
   • QIAprep Spin Miniprep Kit (Qiagen)
   • HindIII restriction enzyme
   • 1 kb plus DNA ladder
   • Low Melting Point Agarose
   • 1X TAE buffer (40 mM Tris Base, 20 mM Acetic Acid and 1mM EDTA)
   • Ethidium Bromide (10mg/mL)
3. General safety notes
   E. coli DH5α is classified as Biosafety Level 1 (BSL1). Microbes in this category pose little to no threat of infection in healthy adults. However, careful manipulation of the microorganism is required.

IMPORTANT all steps in this protocol need to be carried out using aseptic techniques and on ice or 4°C temperatures unless indicated.

2. Protocol

1. From a frozen stock of E. coli DH5α (frozen in 20% glycerol from an overnight culture grown in LB) streak out bacteria for isolation on an LB agar plate. Incubate at 37°C overnight (16-20 hours).
2. Inoculate a single colony into 3 mL of LB broth in a tube. Grow shaking at 210 rpm at 37°C overnight (16-20 hours).
3. Measure the OD600 of the overnight culture. Use the overnight culture to inoculate 100 mL of LB broth in a 1-liter flask to an OD600=0.01. Incubate the culture shaking vigorously (210 rpm) at 37°C monitoring OD600 in the spectrophotometer every 15-20 min, until culture reaches OD600=0.35 (approximately 3 hours).
   NOTE: For transformation to be efficient, bacterial cells need to be at mid-exponential growth phase. The maximum number of cells needs to be 10⁸ cells/mL, which for most strains of E. coli corresponds to OD600=0.4. The use of the spectrophotometer allows to measure the OD600, which allows to determine that the cells are at the appropriate growth stage. If this protocol will be used for other strains of bacteria, calibration to determine the number of colonies forming units at specific OD600 values will be necessary to determine this correlation.
4. Transfer the 50 mL of the culture to each of 2 ice-cold polypropylene centrifuge bottles. Place the bottles on ice for 20 min to cool.
5. Recover the cells by centrifugation at 2700g (4100 rpm in a Sorval GSA rotor) for 10 min at 4°C.
6. Remove the supernatant. Drain away the last traces of media by placing the bottle upside down on a pad or paper towel.
7. Resuspend each bacterial pellet into 30 mL of a CaCl₂-MgCl₂ (80 mM MgCl₂, 20 mM CaCl₂) ice-cold solution. First add 5 mL of the solution, swirl carefully until pellet has dissolved completely and then add the remaining 25 mL of solution.
8. Repeat step 2.4.
9. Repeat step 2.5.
10. If competent cells are going to be directly transformed, resuspend each bacterial pellet into 2 mL of a CaCl₂ (0.1 M) ice-cold solution by swirling the tubes carefully. If the pellet does not get resuspended with this method, resuspend by gently pipetting up and down (avoiding bubble formation).
    Alternatively, the competent cells can be frozen and stored for later use. To prepare frozen stocks of competent cells, resuspend the pellet in 2 ml of a 0.1M CaCl₂ solution containing 10% (v/v) glycerol. This solution needs to be ice-cold. Aliquot cell suspension into ice-cold 1.5 mL polypropylene tubes (160 µl per tube). Freeze competent cells immediately in a dry ice/ethanol bath. Transfer tubes to a -70°C freezer.
11. To transform the CaCl₂-treated cells, transfer 50 µl of competent cells to each of 2 1.5 ml polypropylene tubes. Add the 1 µl (100 pg) of pUC19 plasmid DNA to one of the tubes and leave the second tube without DNA (negative control). Mix gently (avoid bubble formation). Incubate for 30 min on ice.
    NOTE: No more than 50 ng of DNA in a volume of 10 µL or less should be used in the transformation.
12. Transfer the tubes to the heat block and incubate at 42°C for 45 s exactly.
    NOTE: Heat shock is a critical step. Do not exceed temperature or incubation time.
13. Readily transfer tubes to ice. Incubate for 2 min.
14. Add 950 µL of SOC media and incubate the tubes for 1 hour at 37°C to allow the bacteria to recover and express the antibiotic resistant marker encoded in the plasmid.
15. Dilute 10 µL of the cell suspension in 1000 µL in SOC (1/100 dilution) and 100 µL of the cell suspension in 1000 µL in SOC (1/10 dilution). Plate 100 µl of the dilutions, as well as the control, onto selective plates, and spread using a spatula. Usually, plating 100 µL of a 1/100 and 1/10 dilution will yield enough number of colony forming units (cfu) per plate. Ideally, this number should range between 30-300 cfu so that there are enough colonies but separated from each other. However, the number of cfu will depend on the transformation efficiency (see Data Analysis and Results Section).
16. Incubate the plates at 37°C. Transformed colonies should appear in 12-16 hours (this range will depend on the cell strain and selection method). No colonies should grow in the negative control.
17. Count the cfu/plate obtained for the transformation (Table 1).
18. To verify the transformants harbor the pUC19 plasmid, a plasmid preparation and subsequent digestion will be performed. To this end, inoculate a single colony into 3 ml of LB broth in a tube. Grow shaking at 210 rpm at 37°C overnight (16-20 hours).
19. Prepare a plasmid preparation using the QIAprep Spin Miniprep Kit, according to the instructions from the manufacturer.
20. Digest the 1 µg of purified pUC19 with the restriction enzyme HindIII at 37°C for 1 hour.
    NOTE: Any enzyme that cuts in the pUC19 multiple cloning site can be used for this step.

Component	Amount
10X Restriction digest buffer	2.5 µl
Plasmid pUC19	1 µg
HindIII	1 µl
H2O	20.5 µl (to 25 µl)

21. Run a molecular weight ladder, digested pUC19 DNA and the same amount of undigested pUC19 DNA in a 1% agarose gel containing 1 µg/mL ethidium bromide for 1 hour at 95 V.
    NOTE: time and voltage will vary depending on equipment used.
22. Visualize gel under UV light. Compare size of digested and undigested pUC19 DNA (Figure 2) (see Data Analysis and Results Section).
    Proceed with the necessary steps required to verify the transformation according to the goal of each particular transformation experiment.

Figure 2: Digestion of recovered plasmid DNA from transformed DH5α cells. Plasmid DNA was recovered from transformed DH5α cells, digested with HindIII, run in a 1% agarose gel and visualized with a UV source (steps 2.19 to 2.22).

3. Data Analysis and Results

To calculate the transformation efficiency, an indicator of how well the cells took up the extracellular DNA, the colonies obtained in the transformation need to be counted:

Dilution	Cfu
1/100	34
1/10	246

Table 1: Colony forming units (cfu) counted from transformation experiment.

Transformation efficiency (TE) is a measure of the number of cfu resulting from transforming 1 µg of plasmid into a given volume of competent cells. Many parameters affect the transformation efficiency: plasmid size, cell genotype, growth stage during competence preparation, methods of transformation, etc.). When calculating the TE is important to consider what dilution (if any) was performed before plating and incorporate it in the calculation of the total number of cfu. The transformation efficiency (TE) is calculated with the following equation:

First divide the cfu by the µg of DNA, in this example 0.0001µg. Then divide the result by the dilution factor. In this example, a 1/10 dilution was used and 100µL of a 1 ml solution was plated (dilution: 1/10 × 100 µL /1000 µL=0.01).