What Is DNA Transformation

What Is DNA Transformation

Resources » Technical Resource Centers » Gene Technical Resources » Molecular Cloning Center » Plasmid Transformation

Molecular Cloning Strategies

What is PCR

Restriction Enzymes Digestion

DNA Ligation

Plasmid Transformation

DNA Transfection Guide

Transfection Protocol

DNA Transfection Troubleshooting

Ligation Protocol

DNA Ligation Troubleshooting Guide

PCR Protocol, PCR Steps

PCR Troubleshooting Guide

Restriction Digest Protocol

Restriction Digestion Troubleshooting Guide

DNA Ligation Troubleshooting

What is DNA Transformation|Biology

DNA Transformation Protocol

Transformation Troubleshooting Guide

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.

Molecular Cloning Strategies

Since the seminal discoveries of the basic principles underlying molecular cloning.

PCR

PCR stands for polymerase chain reaction and is a method for amplifying DNA.

Restriction Digestion

Restriction digestion also called restriction endonuclease is a process in which DNA is cut at specific sites.

Ligation

Transformation

Plasmid or vector transformation is the process by which exogenous DNA is transferred into the host cell.

Transfection

DNA transfection is the process by which DNA is taken up by eukaryotic cells.

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.

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.

Wrong antibiotic was used or antibiotic concentration was too high

Ensure the correct antibiotic was applied to plates.

Use only concentration recommended by competent cell or antibiotic manufacturer.

Competent cell viability is low

Thaw competent cells on ice and use immediately.

Check expiration date of cells.

Do not re-freeze cells.

Do not vortex cells - gently tap to mix.

Few or no colony transformants

DNA insert encodes protein that is toxic to cells

Use a lower incubation temperature (25–30°C).

Use a cell strain and vector designed for tightly controlled transcription.

Heat-shock incubation too long

Reduce incubation time from 45 to 25 seconds.

Construct is too big

Use electroporation for vectors over 10 kb.

Too much ligation mixture was used for the transformation

Ligation reaction components can inhibit transformation.

Dilute ligation reaction with TE buffer (up to 5 times).

Too much DNA in reaction

Use no more than 1-10 ng of DNA in 5 µl for a 100 µl reaction or in 1-3 µl for a 50 µl reaction.

Low ligation efficiency

Vector insert ratio not optimal. Use a vector:insert molar ratio from 1:1 to 1:10.

Use a DNA concentration of 1-10 µg/ml.

Construct recombined with genomic DNA

Switch to a Rec A- cell strain.

No Plasmid in Colony tranformants

Antibiotic concentration too low

Use antibiotic concentration recommended by manufacturer.

Antibiotic is degraded

Aliquot working volumes of antibiotic and avoid freeze-thaw cycles.

Add antibiotic to liquid plate media after sufficient cooling.

No insert in colony tranformants plasmids

Vector re-ligation

Vector insert ratio not optimal. Use a vector:insert molar ratio from 1:1 to 1:10. Use a DNA concentration of 1-10 µg/ml.

Dephosphorylate DNA with phosphatase to prevent re-ligation.

Sequencing of tranformants plasmid reveals wrong plasmid sequence

Mutations introduced by initial PCR

Use a high-fidelity polymerase.

Inconclusive sequencing artifacts

Repeat sequencing reaction.

REQUEST A QUOTE

EMAIL

[email protected]

PHONE

1-877-436-7274

ONLINE FORM

Online Quote Submission

FAX

1-732-210-0262