**3.2 Transformation of** *E. coli* **cells with plasmid DNA**


#### **3.3 Calculating transformation efficiency of competent cells**

1.Count the number of colony forming units (CFUs) on the LB-agar plate after transformation (see Section 3.2).

**45**

*Growing and Handling of Bacterial Cultures within a Shared Core Facility for Integrated…*

2.Calculate the transformation efficiency (TrEff) in CFUs/μg of DNA using Eq. (1).

*TrEff* <sup>=</sup> # *of CFUs* <sup>×</sup> *Total vol*.*of Transformation* (*L*) \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ *<sup>L</sup> of transformation on plate* <sup>×</sup> *<sup>g</sup> of DNA* (1)

1.Add 5–10 mL of liquid LB to a culture tube and add the appropriate antibiotic to at correct concentration. A good negative control is LB media plus antibiotic without any bacteria inoculated. You should see no growth in this culture after

2.Using a sterile inoculating loop, select a single colony from your LB-agar plate for plasmid purifications and a swipe from 10 to 20 colonies for protein

4.Loosely cover the culture with sterile aluminum foil or a culture tube cap.

5.After incubation, check for growth, which is characterized by a cloudy haze in

6.For overnight cultures, incubate bacterial culture at 30°C for 12–16 h in a shak-

3.Add 500 μL of the overnight culture to 500 μL of 50% glycerol in a 2 mL screw

4.Submerse the glycerol stock tube into liquid nitrogen and store at −80°C. The stock is now stable for years, as long as it is kept at −80°C. Subsequent freeze

5.To recover bacteria from your glycerol stock: open the tube and use a sterile loop, toothpick, or pipette tip to scrape some of the frozen bacteria off of the top. Do not let the glycerol stock thaw. Streak the bacteria onto an LB-agar plate.

<sup>4</sup> For some applications (especially culturing cells in minimal defined media) cultures should never be

overgrown; growing overnight cultures at a reduced temperature, 25–30°C, is suggested.

7.For long-term storage of the bacteria, you can proceed with Section 3.5.

1.Follow Section 3.2 for transforming and plating *E. coli* cells.

2.Follow Section 3.4 for inoculating an overnight culture.

and gently mix.

3.Add the inoculating loop to the liquid LB with antibiotics and swirl.

*DOI: http://dx.doi.org/10.5772/intechopen.81932*

**3.4 Inoculating cultures**

overnight incubation.

expression (Section 3.2).

the media.

ing incubator.4

**3.5 Preparation of a glycerol stock**

top cryogenic vial5

**3.6 Plasmid DNA purification**

<sup>5</sup> Snap top tubes are not recommended.

and thaw cycles reduce shelf life.

1.Preheat the TE Buffer in the incubator at 37°C.

<sup>3</sup> A repeating pipettor or a multichannel pipettor speeds up the aliquoting process greatly. This will minimize the time that the competent cells are manipulated, thus increasing their competency. Expect competency of ca. 107 –108 CFU/μg of plasmid DNA.

*Growing and Handling of Bacterial Cultures within a Shared Core Facility for Integrated… DOI: http://dx.doi.org/10.5772/intechopen.81932*

2.Calculate the transformation efficiency (TrEff) in CFUs/μg of DNA using Eq. (1).

*TrEff* <sup>=</sup> # *of CFUs* <sup>×</sup> *Total vol*.*of Transformation* (*L*) \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ *<sup>L</sup> of transformation on plate* <sup>×</sup> *<sup>g</sup> of DNA* (1)

## **3.4 Inoculating cultures**

*Growing and Handling of Bacterial Cultures*

7.Spin for 10 min at 6000× g at 4°C.

DMSO.

tubes.3

20–30 min).

trifuge tube.

6.Incubate the cell suspension on ice for at least 10 min.

9.Incubate the cell suspension on ice for at least 10 min.

12.At −80°C the cells will be competent for at least 6 months.

**3.2 Transformation of** *E. coli* **cells with plasmid DNA**

ally warm to 37°C in an incubator.

6.Heat shock each tube at 42°C for 45–60 s.

7.Put the tubes back on ice for 2 min.

10 cm LB-agar plate.

10.Incubate plates at 37°C overnight.

transformation (see Section 3.2).

–108

8.Gently resuspend the pellet in 9.4 mL ice-cold CC buffer and add 0.7 mL

10.Distribute the cell suspension in 50–200 μL aliquots in 1.5-mL microcentrifuge

11.Flash freeze the cell suspension in liquid nitrogen and store the tubes at −80°C.

1.Take competent cells out of −80°C and thaw on ice (approximately

2.For each transformation, remove two LB-agar plates (containing the appropriate antibiotic) from storage at 4°C and warm to room temperature; option-

3.Mix 10–100 pg. DNA into 20–50 μL of competent cells in a 1.5 mL microcen-

4.Gently mix by flicking the bottom of the tube with your finger a few times.

8.Add 1 mL of LB medium (without antibiotic) to the bacteria and grow at

9.Plate 50 μL of the transformed cells onto one of the 10 cm LB-agar plate containing the appropriate antibiotic and the remaining 950 μL onto the second

1.Count the number of colony forming units (CFUs) on the LB-agar plate after

<sup>3</sup> A repeating pipettor or a multichannel pipettor speeds up the aliquoting process greatly. This will minimize the time that the competent cells are manipulated, thus increasing their competency. Expect

5.Incubate the competent cell/DNA mixture on ice for 20–30 min.

37°C and 250 rpm in a shaking incubator for 45 min.

**3.3 Calculating transformation efficiency of competent cells**

CFU/μg of plasmid DNA.

**44**

competency of ca. 107

