Team:PrepaTec GarzaSadaMx/Protocols

Protocol Competent cells:

As shown in the paper made by Anh-Hue T. Tu (2), to make competent cells you should perform the following steps: Inoculate 5 mL of LB media with E. Coli and grow overnight at 37°C on an incubator. Take 1 mL of the culture growth overnight into a sterile Erlenmeyer flask with 100 mL of LB broth. To make sure that you’ll get most of the cells in the mid-log growth phase (5x107 cells/mL), you should take the culture into a 37°C water bath. This process should take 2 to 4 hours. The growth rate is determined by the optical density of the sample. Remove 1-ml aliquot at various times and read the optical density at 600 nm wavelength, using the spectrophotometer. The cells should be on this stage because it has been shown by several studies that in the mid-log growth phase it’s when the cells are transformed efficiently. When the step 3 is done, the cells are in the mid-log growth phase. Cool down the culture on ice for 10 minutes (The ice is at 4°C). Spin the cell suspension at 4000 rpm in a centrifuge for 5 minutes at 4°C Discard the supernatant. Resuspend the cells in half the volume of the original culture (50 mL of LB broth) with ice-cold sterile 10 mL of CaCl2. Place the cell suspension in an ice bath for 10 minutes. Centrifuge the suspension at 4000 rpm for 5 minutes at 4°C. Discard de supernatant. At the end of the suspension and resuspension, what it will be got is the pellet of cells. This pellet, at 4°C, should be next be putted on a hot water bath at 42°C. This will create pores on the membrane of the cells, allowing the DNA molecules to get into the E. Coli. Carefully, resuspend the cells in 10 mL of sterile ice-cold CaCl2 using precooled pipettes. The cells will remain competent for up to 24 hours at 4°C. The transformation efficiency increases four-to-six fold during this time. For long-term storage, cover a tube with aluminum, where the cells should be stored, and put in a tank with liquid nitrogen at -80°C. Depending on the strained used, the E.Coli cells will remain competent to take up DNA for as long as 6 months, making a competency test each time an aliquot is removed from storage to be used for transformation.

Transformation of cells.

Summary:

The transformation is a process by which cells (in this case E. coli) are able to identify DNA present in the surrounding area. In order for this to take place, the E. coli has to be in a competent state.1 Some bacteria are naturally in this state, however, this is no the case of the E. coli, thus this state is achieved by following the previous process. In this state, the bacteria presents some alterations in its cellular wall and membrane, which now will allow the entrance of nucleic acids to the cell.1 The entrance of foreign DNA in the form of a plasmid –either in its circular form or in its supercoiled form- to the cell is what’s called transformation.1

After the cell is transformed, it has to go through the step of selection. This final step is very important, because after doing this, it’s possible that some of the bacteria didn’t take up the plasmid and therefore, in order to identify the transformed bacteria, the DNA in the plasmid has a marker.1 The marker consist in making the transformed cells resistant to the selective media. In this experiment, the media where the E. coli cells are going to be tested will contain ampicillin, and consequently only the cells with the plasmid –which will make them ampicillin resistant- are going to survive.1 By doing this, we ensure that the cells that we harvest are the ones that contain the mentioned plasmid, as they were able to develop in the selective media.1

Materials:

Luria broth (LB) SOC medium LB plates containing ampicillin Filter-sterilized antibiotic solution (ampicillin) E. coli K12 derivatives (TB1, JM109) Plasmid vector: pPH219 1x Tris-EDTA (TE) buffer pH 8.0 (10 mM Tris Cl, 1 mM EDTA pH 8.0) Sterile 60 mM cold CaCl2 solution (60 mM CaCl2, 15% glycerol, 10 mM piperazine-N,N’-bis(2-hydroxypropanesulfonic acid) (PIPES), pH 7) Pipetters (1 to 10 μl and 10 to 200 μl range) Sterile pipette tips Sterile 50-ml glass or polypropylene tubes with caps 250-ml sterile Erlenmeyer flask with cap 5-ml glass or plastic pipettes precooled in refrigerator Thermometer (0 to 100°C) Permanent marker for labeling Clock or watch for timing 0.2-mm filter (Fisher Scientific)

       Styrofoam bucket with crushed ice  

37°C incubator Water bath shaker set at 37°C 42°C water bath (using a thermometer ensure the temperature is exactly 42°C for the 2 minutes of heat shock) Centrifuge Roller drum in a 37°C incubator

Protocol:

Add 10 to 40 ng (10 to 25 ml volume) of DNA to 250 ml of competent cells. Concentrated stock DNA can be diluted using sterile 1x TE buffer. This can be done to prevent a decrease in efficiency resulting from high concentration of DNA. Besides the DNA tube, additional tubes should be set up as listed below: In order to keep a tube denominated ¨control¨. This tube will contain 1x TE buffer in the same volume of DNA used in step 12. For the remainder of the experiment, this tube will also be submitted to the next steps. If competent cells were kept at -70℃, then it’s necessary to ensure that these remain able to take up DNA. The thawed cells are incubated with 10 ng of a control plasmid such as pBR322. The number of transformants per microgram of DNA will be calculated and should typically yield from 106 to 108 colonies/mg DNA for E. coli MC1061 and DH1 cells. Incubate the mixture on ice for 30 minutes. For the next 2 minutes, the mixture must be in a 42oC water bath. Incubate on ice for 5 minutes. As a next step, add 1.0 ml of SOC medium to each tube and incubate at 37oC for 1 hour in a roller drum (250 rpm). This will allow cells to express the antibiotic resistance marker, making it possible for them to survive in the selective media. Spread 50 to 100mL of cells on the selective media mentioned above. Store the remaining cells at 4oC. The E. coli cells from the control tube will be planted on selective medium and nonselective medium. In the first case, there should be no growth present, as it is a way of ensuring that, in absence of the DNA added, there would be no resistance to ampicillin, and thus the cells do not grow. On the other hand, the second plating offers viable cells that can develop in the absence of a selective medium. Cells that have to be tested in order to prove that they are still competent should be plated on LB agar with ampicillin (50 mg/ml final concentration). If the E. coli cells are able to survive, then it’s possible to conclude that transformation efficiency hasn’t decreased. Incubate all plates overnight at 37oC (agar side up). Count the number of colonies.