The+Transformation+Page

// Transformation

Purpose of the Transformation: The transformation step in the experiment is extremely important; it's performed to put our ligated DNA into cells. Two transformations are performed in this experiment. The first one involves DH5α and is used because we need to be sure that we have ligated plasmid containing the Bli-1 gene. DH5α is used because it is very competent and takes on plasmids very easily. The second transformation involves HT115 and is crucial because the transformed HT115 is fed to the worms to silence the Bli-1 gene. Procedure for Making Competent Cells 1. Place the cell culture on ice for 10 minutes 2. Pellet the cells at 2,500x g for 6 minutes at 0-4°C 3. Discard the supernatant and re-suspend the cells in 5 ml of cold 1X Wash Buffer 4. Repeat step 2 5. Discard the supernatant and re-suspend the cells in 5 ml of cold 1X Competent Buffer 6. Put 100μl of the cells into a tube and put on ice.

Procedure for the Transformation of the DH5α and the HT115 //

1. Put 100μl of competent cells in a tube 2. Add 5μl from the ligation into the tube 3. Gently mix the tube and incubate the tube on ice for 60 minutes 4. Add 400μl of LB to the tube 5. Incubate the tube for 60 minutes at 37°C 6. Spread 250μl from the tube on to one of the AMP plates that was pre-warmed to 37°C

For our experiment, we chose to transform 4 tubes of the DH5α with our ligation to be sure that we could obtain enough samples of the successfully transformed cells. We transformed a 5th tube with PUC19 to use as a control to make sure that the cells were being transformed. We chose PUC19 because we know that DH5α takes in the plasmid easily from previous experiments. For the others, we transformed the DH5α with the ligated L4440 and the PCR product in order to make cells that contained the Bli-1 gene. From these 5 tubes we made 8 plates on which to grow the cells.

A total of 11 colonies grew on the 8 plates. The PUC19 plates did not have any growth which was concerning but 2 of the DH5α plates had growth. From these plates we split each of these individual colonies in half and with one half of them we ran a single-colony PCR to verify that the cells were transformed with the L4440 that contained the Bli-1 gene. With the other half of the colonies, made 11 LB broths with AMP. The broths contained AMP because the L4440 contains the AMP resistance gene, meaning that only the successfully transformed cells would grow in the broth. While the colonies should all have AMP resistance because they were grown on the AMP plates, the AMP broth ensures that no satellite colonies make it into the broth. In the single-colony  PCR, we are taking advantage of the T7 promoters on the L4440 to amplify whether or not the gene of interest was ligated into the plasmid. The T7 promoters are located on the plasmid outside of where the Bli-1 gene should have been inserted. After this PCR finished we ran a gel to find whether or not the Bli-1 gene was in the transformed cells. The results from the gel are below.

The 11 broths were left to grow in a shaking water bath. All of them successfully grew so we then performed a mini-prep on all of them to isolate the DNA. Unfortunately, our experiment concluded after this point. Had we continued, we would have used the information from our PCR gel to determine which of the colonies contained the Bli-1 gene. Our current gels (shown below) do not provide us with much information though, but due to the lack of bands, it seems that the PCR was unsuccessful because the cells were not lysed so there was no DNA to be amplified. Had we continued, it is likely that we would have used the DNA from our mini-preps to run another PCR to find which colonies contained the Bli-1 gene.  We would have then used the corresponding mini-prep from the same colony to transform the HT115. The transformed HT115 would then be fed to the worms. Once the worms were fed the HT115, the Bli-1 gene would be silenced and the worms would have blisters.

The Single-Colony PCR Gels: