February 26th to April 8th

I successfully performed an RNA extraction with whole seedlings, followed by DNA free treatment. Using this freshly extracted RNA, I performed RT-PCR with the new primers I developed on the 5' end for each gene, in addition to the original 5' primer. This was performed as a control to determine whether the genes were actually expressed in the seedling. When I analyzed the results via gel electrophoresis there were bands for each different gene, so we now knew that these genes are expressed in the seedling.
Next, I extracted fresh RNA from both root tissue and root tip tissue, followed by DNA free treatment. I again performed RT-PCR on this freshly extracted RNA with the new primers I developed on the 5' end for each gene, in addition to the original 5' primer. The results were visualized on agarose gel and there were bands showing for each gene. These bands were excised from the gel and stored in -20 C.
Then I attempted the generacer kit using the freshly extracted root RNA. I again followed the exact specifications outlined in the protocol provided with the kit to amplify the cDNA ends. The 5' primers used were actually the reverse complement of the original primer. These primers were chosen because the fragments obtained in the RT-PCR were longer then expected, leading me to believe that the actually regions I was after were longer than expected. I was hoping using primers that were further upstream would allow a capture of the 5' end. I also used the newly created 5' primers for cDNA amplification. For the 3' end, I used the original 3' primer as well as the two newly created primers. The only bands that were visualized on the agarose gel were for the newly created 5' primers, but these bands were very smeary. Every other primer used did not have any results.
Next, I nanodropped the race ready cDNA that I was using to determine the concentration, which was 440 ng/ul. Then, I diluted the cDNA to a final concentration of 50 ng/ul and performed the cDNA amplification again. I used all of the primers, both the original 5' and 3' primers, the newly created 5' and 3' primers, and the reverse complement of the 5' primers. Now, visualization showed no bands at all.
Since I have not had any success at all with the generacer kit I am going to attempt oligo capping, which is very similar to RACE. Hopefully, it will allow sequencing of the 5' ends of these genes. I will actually be performing this protocol for all of the transcripts found in 3 day old root tips, 18 day old roots, and 3 day old roots. I am working on getting everything ready for the protocol and hope to be able to begin by next week.