Bacterial Transformation Protocols

Transformation is a key process in molecular cloning, by which multiple copies of recombinant DNA molecules are produced. The ability to take up free, extracellular genetic material is the prerequisite for bacterial competent cells to undergo transformation. The objective is to obtain the replication of sequence of interest of a recombinant plasmid.

Figure 1. Transformation

Before starting transformation:

• Prepare LB agar plates and allow to set. If pre-poured plates are being used, ensure the plates are warmed to 37 °C.
• Depending on the antibiotic marker present in the plasmid DNA, incorporate appropriate antibiotic in the LB agar.
• If blue/white screening for recombinants is required, incorporate 1 mM IPTG, 300 µg/mL S-Gal or 40 µg/mL X-Gal and 500 µg/mL ferric ammonium citrate in the LB agar.
• If electrocompetent cells are being used, place the electroporation chamber on ice.
• Heat the water bath to 37 °C.
• Warm the sterile SOC medium to room temperature (or 20-25 °C in water bath).

Protocol for transformation using chemically competent cells

Materials required but not provided:

Reagents	Equipment
SOC medium (Product No. S1797)	Shaker incubator (37 °C)
LB agar EZMix™ (Product No. L7533)	Cabinet incubator (37 °C)
Appropriate selection antibiotic	Heated water bath (37 °C)
IPTG (Isopropyl-β-D-thiogalactoside) (Product No. I6758)	15 mL polypropylene culture tubes (sterile)
X-gal (Product No. B9146)	Culture dishes
S-Gal™ (Product No. S9811)	Lazy-L sterile spreaders (Product No. Z376779)
Ferric Ammonium citrate (Product No. F5879)

Standard Transformation Protocol

1. Transfer the required number of tubes from -70° C freezer to wet ice. Include an extra tube for control DNA, if desired
2. Allow the cells to thaw for 5 minutes. Gently tap the tubes multiple times to obtain uniform suspension
   1. For control: Add 1 µL (10 ng) pUC19 control DNA to one tube. Mix by gentle tapping and place on ice
   2. Add 1 ng to 50 ng of purified plasmid DNA directly to cells in rest of the tubes. Mix by gentle tapping and place on ice
3. Incubate the cells on ice for 30 minutes
4. Transfer the cells to 37° C water bath for exactly 45 seconds
5. Transfer the cells to ice for 2 minutes
6. Add SOC medium to each tube. Transfer the cells to sterile polypropylene tubes and loosen the caps to facilitate aeration of the cultures
7. Incubates the cells on shaker incubator (225-250 rpm) at 37° C for 1 hour
8. Pipette 10-100 µL of each transformed cell suspension onto LB agar plates with selection antibiotic and spread it using sterile spreader
9. Incubate plates at 37° C overnight
10. Select colony (colonies) and culture as needed
11. Isolate the plasmid DNA from each culture
12. Culture the preferred clones
13. Digest the plasmid DNA using restriction enzymes; separate by gel electrophoresis

Protocol for transformation using Electrocompetent cells

Materials required but not provided:

Reagents	Equipment
SOC medium (Product No. S1797)	Shaker incubator (37 °C)
LB agar EZMix™ (Product No. L7533)	Cabinet incubator (37 °C)
Appropriate selection antibiotic	Heated water bath (37 °C)
IPTG (Isopropyl-β-D-thiogalactoside) (Product No. I6758)	15 mL polypropylene culture tubes (sterile)
X-gal (Product No. B9146)	Culture dishes
S-Gal™ (Product No. S9811)	Lazy-L sterile spreaders (Product No. Z376779)
Ferric Ammonium citrate (Product No. F5879)	Culture dishes

Standard Transformation Protocol

1. Transfer the required number of tubes from -70° C freezer to wet ice. Include an extra tube for control DNA, if desired.
2. Allow the cells to thaw for 5 minutes. Gently tap the tubes multiple times to obtain uniform suspension.
3. Transfer SOC medium to culture tubes, one for each transformation reaction, and leave at room temperature.
   
   (The volume of SOC medium depends on the volume of cells that will be added in the next step. The final volume with the competent cells and SOC medium should be 1000 µL)
4. Place 1 mm standard cuvettes and sterile microcentrifuge tubes on ice, one for each transformation reaction.
5. Transfer the competent cells to chilled microcentrifuge tubes. Use 40 µL of cells from 80 µL package and 50 µL of cells from 100 µL package.
   1. For control: Add 1 µL of 1 to 5 diluted pUC19 control DNA to one tube.
   2. Prepare 1 to 5 fold dilution of DNA or ligation mix in TE buffer. Add to microfuge tubes.
6. Pipette the DNA and cells mixture into chilled 1 mm cuvette.
7. Set electroporator to a field strength of 25 kV/cm for 6 ms and treat the cells.
8. Remove cells from cuvettes and add to tubes containing SOC medium.
9. Incubate the cells on shaker incubator (225-250 rpm) at 37° C for 1 hour
10. Pipette 10-100 µL of each transformed cell suspension onto LB agar plates with selection antibiotic and spread it using sterile spreader.
11. Incubate plates at 37° C overnight.
12. Select a colony and culture as needed.
13. Isolate the plasmid DNA from each culture.
14. Digest the plasmid DNA using restriction enzymes; separate by gel electrophoresis.
15. Culture the preferred clones.

Important tips for optimization of transformation process:

• Confirm that the cells are still frozen and dry ice is still present upon receipt.
• For maximum transformation efficiency, use high quality DNA sample free of phenol, ethanol, proteins, salts or detergents. When using electrocompetent cells, high salt content in DNA will result in arcing at high voltage which may damage the sample and the equipment.
• DNA in ligation reactions containing high quality reagents may be used for transformation. The DNA ligase need not be inactivated.
• Keep the competent cells on ice at all times; prevent accidental warming of the cells.
• Tap the tubes gently to obtain uniform suspension of cells. Do not mix by using vortex or pipette.
• Warm LB agar plates to 37 °C for optimal colony growth.
• Electroporator settings for transformation using electrocompetent cells:
  • BTX Model ECM 630: HV mode, 2.5 kV, 25 µF, 100 Ω, 1 mm cuvette
  • BioRad Gene Pulser: 2.5 kV, 25 µF, 100 Ω, 1 mm cuvette

References

1. Sambrook J, Fritsch E, Maniatis T. 1989. Molecular cloning: a laboratory manual.. New York: Cold spring harbor laboratory press.

2. Dubnau D. 1999. DNA Uptake in Bacteria. Annu. Rev. Microbiol.. 53(1):217-244. https://doi.org/10.1146/annurev.micro.53.1.217

3. Lorenz MG, Wackernagel W. 1994. Bacterial gene transfer by natural genetic transformation in the environment.. 58(3):563-602. https://doi.org/10.1128/mmbr.58.3.563-602.1994