Site Directed Mutagenesis:

In order to be able to create a larger gap in the YTM1 gene in the plasmid pBM258T/YTM1, I attempted to create a second SnaBI restriction site by site directed mutagenesis. I talked to both Carol and Jelena about their previous site directed mutagenesis experiments, and modeled my reaction conditions after them. My primers were 39 nucleotides in length and had a Tm of 79 deg C—good conditions for creating two mutations separated by only four bases. I tried many different PCR reaction conditions, which are outlined in table 2. For all of the various reactions, I followed the basic procedure outlined in the Stratagene kit protocol. I mutated my plasmid using PCR, digested the parent plasmid with DpnI and transformed into competent DH5-a cells using electroporation. Because I was working with a large plasmid (9.6kb) I used the high fidelity Pfu Ultra DNA polymerase for all of the reactions.

1. The first set of site directed mutagenesis reactions yielded no transformants. This may have been due to the age of the competent DH5-a cells used. Also, I did not purify the DNA in the reactions after digestion with DpnI.

2. For the second set of reactions I purified the DNA after digestion with DpnI by precipitating and washing with ethanol. The ethanol purification helps the transformation because it gets rid of salts that are in the final site directed mutagenesis solution with the DNA. Electroporation does not work as well when salts are present. I did two different transformations: the first transformation was into the older competent cells and yielded only one transformant. However, when I repeated the transformation with new competent cells, I got thousands of colonies.

3. Again, I had purified the DNA by precipitating and washing with ethanol for the third set of reactions. I also used the new competent cells. Although I saw a large number of transformants on one of my plates, I also saw an even greater number of colonies growing on the negative control that contained no DNA. I need to repeat this transformation.

I began screening through the transformants. If two SnaBI sites are present in the plasmid, I expect to see two bands when I run the digested plasmids on an agarose gel: 8759bp and 881bp.

1. The first transformation of the second set of reactions had yielded one transformant. I mini prepped the plasmid with the BioRad kit and digested with SnaBI. Analysis of the digest on a 1% agarose gel showed only one band at the same size as the control pBM258T/YTM1 digest (9640bp). A second SnaBI site had not been created.

2. I picked 12 transformants from the second transformation of the second set of reactions and mini prepped using the regular protocol and not the kit. I digested with SnabI and ran the digests on a 1% agarose gel. I saw only one band at about 9640bp for each of the digests. This is shown in figure 3. I had trouble interpreting the agarose gels at first because there was a lot of degraded RNA present in the bottom of the gel. However, it was pretty clear that only one band was present. No bands were seen around 900bp. Again there was no second SnaBI site formed. I picked another 24 transformants, mini prepped with the regular protocol, digested with SnaBI, and ran the digests on a 1% agarose gel. As before, I only saw one band. In order to make sure that I was seeing only one band on the gel, I chose six of the transformants and mini prepped them with the BioRad kit. I digested with SnaBI and ran the digests on a gel. It was very clear this time that there was only one band at 9640kb. This is shown in figure 4.

I am continuing to screen through transformants from the second set of site directed mutagenesis reactions.

Figure 3: Example of Site Directed Mutagenesis Results- Regular Mini Prep Protocol


Figure 4: Site Directed Mutagenesis Results- BioRad Mini Prep Kit