General Transformation

by Tannon Yu

Contents

Background

Bacterial transformation is the biological phenomenom where a bacterium uptakes free-floating plasmid DNA from its environment and uses it as its own. Such a process generally only occurs when the bacterium is in a competent state, which results from natural environmental stresses or is artificially induced via in vitro means.

Competent cells used in the Şen Lab are generated via a rubidium chloride protocol, ensuring high transformation efficiency, and are always stored at −80 °C. Tradition dictates that conventional transformation require both heat shock and outgrowth steps. Heat shocking the competent cells is believed to open the membrane barriers of the bacteria, allowing more efficient intake of exogenous plasmid DNA into the cells. Subsequent outgrowth in media helps the cells recover from the high stress of heat shocking.

The competent cells in our laboratory are efficient enough to successfully perform transformation without the need for conventional heat shock steps (3a–b). For plasmids containing ampicillin (or carbenicillin) resistance, the protocol can be shortened even further by skipping the outgrowth steps (3c–d) as well. Any other antibiotic resistances, such as kanamycin or chloramphenicol, require the outgrowth steps but can still skip heat shocking.

For cases where the highest level of transformation efficiency is desired (such as in site-directed mutagenesis), it is recommended to include both heat shocking and outgrowth steps.

Protocol

1. Thaw competent cells on ice for approximately 5–10 minutes. Never thaw by hand.
2. Add 100–200 ng of plasmid to the competent cells tube. Mix by gently tapping the side of the tube. Do NOT vortex or pipette the mix.
3. Incubate on ice for 5–10 minutes. To increase transformation efficiency, the following optional* steps can be performed. Otherwise, it is safe to proceed directly to step 4.
   1. Heat shock the cells in a 42 °C water bath for 40–45 seconds.
   2. Immediately place cells on ice for 2 minutes.
   3. Add 200–300 μL of LB media to the tube.
   4. Incubate the cells at 37 °C while shaking at 250 rpm for 45 minutes.
4. Spread contents of the entire transformation tube on an LB media plate containing the appropriate antibiotic. Incubate at 37 °C for 16–18 hours.

* NOTE: Only plasmids resistant to ampicillin (carbenicillin) can skip the outgrowth steps (3c–d). Any other antibiotic resistances require outgrowth.