What is DNA transformation
Plasmid or vector transformation is the process by which exogenous DNA is transferred into the host cell. Transformation usually implies uptake of DNA into bacterial, yeast or plant cells, while transfection is a term usually reserved for mammalian cells. Typically the method for transformation of a DNA construct into a host cell is chemical transformation, electroporation or particle bombardment. In chemical transformation, cell are made competent (able to take up exogenous DNA) by treatment with divalent cations such as calcium chloride, which make the bacterial cell wall more permeable to DNA. Heat shock is used to temporarily form pores in the cell membrane, allowing transfer of the exogenous DNA into the cell. In electroporation, a short electrical pulse is used to make the bacterial cell temporarily permeable. Particle bombardment, is typically used for the transformation of plant cells. Gold or tungsten particles are coated with the DNA construct and physically forced into the cell by gene gun.
DNA transformation protocol
- Thaw all reagents completely on ice.
- Add 1 µL of ligation reaction to thawed competent cells.
- Gently mix by tapping tube of competent cells.
- Incubate reaction on ice for 30 minutes.
- Heat shock the competent cell mixture by incubation for 30 to 60 seconds in a 42°C water bath.
- Incubate tubes on ice for 2 minutes.
- Add 250 to 500 µL of SOC or LB media.
- Incubate at 37°C and shake at 250 rpm.
- Warm selection plates to 37°C.
- Spread 10, 50, and 100 µL of transformed cells on selection plates.
- Incubate plates at 30°C overnight.
Component |
Final concentration/amount |
---|---|
Competent cells |
to 50 µL |
Ligation reaction |
1-5 µL |
SOC or LB media |
950 ml |
Transformation troubleshooting guide
A successful plasmid transformation is dependent on a number variables including antibiotic concentration, construct size and concentration, and ligation efficiency. Use the troubleshooting guide below to optimize your transformation reactions or get your desired gene in the vector you want the easy way with GenEZ™ ORF clones. Start with a search for your gene.
Problem | Causes | Solutions |
---|---|---|
Wrong antibiotic was used or antibiotic concentration was too high |
|
|
Competent cell viability is low |
|
|
Few or no colony transformants |
DNA insert encodes protein that is toxic to cells |
|
Heat-shock incubation too long |
|
|
Construct is too big |
|
|
Too much ligation mixture was used for the transformation | Ligation reaction components can inhibit transformation.
|
|
Too much DNA in reaction |
|
|
Low ligation efficiency |
|
|
Construct recombined with genomic DNA |
|
|
No Plasmid in Colony tranformants |
Antibiotic concentration too low |
|
Antibiotic is degraded |
|
|
No insert in colony tranformants plasmids |
Vector re-ligation |
|
Sequencing of tranformants plasmid reveals wrong plasmid sequence |
DNA insert encodes protein that is toxic to cells |
|
Mutations introduced by initial PCR |
|
|
Inconclusive sequencing artifacts |
|