The overall goal of this procedure is to generate competent bacteria rapidly and conveniently on the same day. They are needed for transformation. This is accomplished by first plating the bacteria on LB agar and incubating them for four to six hours.
Next, the bacterial lawn is carefully harvested from the surface of the agar, and the pellet is resuspended in sterile cold water or buffer. Then the bacterial pellet is washed three times at four degrees Celsius and resuspended in 40 microliters. Finally, the competent cells are electroporated in the presence of DNA.
Ultimately, results can be obtained that show levels of bacterial transformation comparable to those obtained using the traditional method that requires considerable equipment, large volumes of bacterial cultures and sterile buffers. The main advantage of this technique over existing methods is that fresh, competent bacteria can be prepared virtually from any laboratory strain the same day that the transformation is planned without the need for large centrifuges, rotors, or leaders of sterile buffers and cultures Generally, individuals new to this method will not struggle much because this is just a shortcut variation of techniques commonly employed in bacteriology laboratories. The originator of this protocol cannot be identified as it has been used and optimized by numerous researchers over time.
The method presented here has been used in our laboratories for nearly two decades, and it is our goal to share this useful protocol with our peers. Visual demonstration of this method is important for one step. In particular, the collection of bacteria from the lawn on the auger because it takes an experience eye to evaluate the growth on the plate.
And this is best visualized. Demonstrating the procedure is Ms.Theresa Brooks, a research assistant from the laboratory of Dr.Te Koski at University of Alberta in Edmonton, Canada. I to begin bacterial culture preparation in the late afternoon, use a small amount of e coli or v coray to inoculate one to five milliliters of autoclave LB broth in sterile bo silicate glass test tubes.
Set the inoculated test tubes in a roller drum and incubate overnight at 37 degrees Celsius and high speed. Prepare a cell spreader in the shape of a hockey stick from a glass rod by heating the middle of the rod in the flame of a bunsen burner until the glass softens. Then use tweezers or pliers to gently direct the bend into a 135 degree angle.
Heat the rod once more at the midpoint between the end and the first bend and bend it at a 45 degree angle inwards. Prepare lb aerates with and without antibiotics and store at four degrees Celsius. The next morning.
Plate 100 microliters of overnight culture onto each LB agar plate. Using the cell spreader, incubate the plate at 37 degrees Celsius for four to six hours or until a thin lawn of bacterial growth becomes visible. Using an inoculating loop harvest an about two millimeter diameter bacterial mass from the plate and resus suspend it in one milliliter of ice cold.
Two millimolar calcium chloride. If using v coray or sterile doubly distilled water for e coli using a refrigerated centrifuge set at four degrees Celsius, spin the suspension for five minutes at 5, 000 Gs.Discard the supernatant and repeat the wash two more times. After removing the final supernatant resus, suspend the pellet in 40 microliters of calcium chloride or doubly distilled water and keep on ice.
To carry out a transformation, add up to one microgram of plasma DNA to the 40 microliter bacterial suspension, and transfer the mixture into a pre chilled sterile 0.2 centimeter gap. Vete, insert the vete into the electroporation chamber of the pulse control module and ate at 1.8 kilovolts and 25 micro phs. The time constant should be about 5.0 milliseconds and no arcing should occur.
Quickly resuspend the cells in one milliliter of LB broth and transfer into a previously autoclave broil glass test tube. Allow the cells to recover by incubating under aerated growth conditions at 37 degrees Celsius for 30 minutes without antibiotic selection. Plate the bacteria onto LB ager plates with antibiotic and incubate at 37 degrees Celsius overnight.
As shown here, we carried out a set of transformations with e coli and V co array to compare the transformation efficiency of our rapid methodology with an adaptation of the traditional method originally described by doer et al for the preparation of electro competent bacteria. The transformant yield was within the 10 of the sixth to 10 of the seventh CFUs per microgram of DNA range in three, replicate experiments for e coli and v coray. Using the traditional method for electro competence.
Cell preparation, transformant yield appeared decreased tenfold to 10, to the fifth, to 10 to the sixth CFUs per microgram of DNA in three replicate experiments for e coli and v coray using the rapid method. For this reason, we determined the percentage of transformed bacteria by dividing transformed CFUs by the number of CFUs recovered from mock transformations from otherwise identical batches of competent cells. The percentage of transformed bacteria was within one log.
Regardless of the method of preparation and species transformed. These results suggest that the efficiency of the rapid method is comparable to that of the traditional lengthier procedure of preparing competent cells, and that the representative strains of ieo, coray, and esia coli are equally amenable to the rapid procedure. Once master, this technique can be done within five to six hours, performed properly.
While attempting this procedure, it is important to remember to harvest the bacteria during their logarithmic phase of growth, wash them in sterile, freshly prepared and pre chilled buffer water solution and keeping them cold until resuspended in LB past the electroporation. After watching this video, you should have a good understanding on how to rapidly generate electro competent bacterial strain specific cells in your laboratory by cultivating them on inaugural lawn, collecting them during logarithmic phase of growth, and then washing them in cold buffer in preparation for electroporation.
