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.

January 15th to February 25th

To begin, I checked the integrity of the RNA that I was using in the generacer kit. Everything checked out great and the RNA was of sufficient quantity and quality to use. I began the generacer kit using root RNA and the control HeLa RNA provided in the kit. Following each step in the protocol, I analyzed the integrity of the RNA via agarose gel electrophoresis. This allowed a confirmation that the product was maintained throughout the reaction. I finished the generacer kit on 1-28-11. On 1-31-11, I amplified the ends of the cDNA that was generated following the exact specifications provided in the protocol for the generacer kit. I also included all of the negative controls that were recommended in the kit. The first attempt at visualization via electrophoresis was not successful. A few bands were showing but they were very faint. Next, I concentrated the PCR products that were remaining (40 uL) and reran the gels. The HeLa control reactions were successful but there were no bands showing for either the 5' or 3' end of any of the WRKY genes.
In an attempt to determine why the bands were not showing up for any of the target genes, I performed a one step RT PCR reaction with both root tip and root RNA. This would determine if the three genes were actually expressed in the target tissues. The resulting bands were very faint and led to the conclusion that the genes were not highly expressed in root tips or root tissues.
To circumvent this problem of expression level in these tissues, I will extract RNA from whole seedlings that were allowed to germinate for 5 days. I will then perform the generacer protocol with this RNA. Additionally, I developed 2 new primers for both the 5' end and the 3' end of each gene, which have different binding sites and may give more specific results. I will use all three primers that I have for each gene when I analyze the products generated from the generacer kit.
Hopefully, with these changes I will be able to sequence the 5' and 3' ends of these genes. I will start next week by extracting the RNA and then proceed with the generacer kit.

December 21st to January 14th

I finally recieved the new chemicals that I need to perform the controls. As I perform the control reaction included with the Generacer kit, I will also simultaneously perform the experimental reaction. To determine where the problem is occurring during the experiment, I will run tests on my sample following each step. This should allow me to pinpoint the source of error and from there I can devise a method to overcome any problems that are occurring. I have set everything up to begin this phase of the project next week and will update my status within 2 weeks.

November 21st to December 20th

After purifying the plasmid DNA, I performed PCR to excise the target DNA fragments. Unfortunately, the results I obtained were very unusual. There were multiple bands observed from each miniprep sample. There were three bands showing for each different sample and the bands were the same for each different sample. I am not sure what went wrong and neither is Dr. Schlueter. I am still waiting on the necessary supplies to perform the experiment and the controls together. Hopefully, this will allow us to pinpoint the source of the error.

October 31st to November 20th

In order to obtain bands for the 5' end we are ordering the pfx taq that is recommended in the generacer kit and I will be running a control on this as soon as we receive it. I repeated the cloning reaction on the 3' ends obtained in PCR, but was unable to do the control because we don't have kanamycin. We were using ampicillin screening on the colonies. I cloned the PCR products I did have and a few colonies grew on each plate. To purify the colonies I performed a mini prep, using two different colonies from each plate. I nanodropped the results of the mini prep and do have ample DNA. Using the purified plasmids, I am now performing PCR to excise the DNA sequences in order to use them in sequencing.
Hopefully over the next month I will be able to perform sequencing on the 3' ends and overcome the challenges on the 5' ends. Unfortunately, oligo capping doesn't always work but I am still hopeful that we can find a way to get results with this procedure.

October 10th to October 30th

This has been a busy few weeks for me. I began by cloning the extracted bands for the 3' ends of each gene using a TOPO TA cloning kit and TOP10 chemically competent cells. Nothing grew on these plates so I am going to run a positive control and replate the excised bands at the end of this week hopefully.
I used the newly prepared cDNA to run PCR again on the 5' ends of each gene but still no bands were visible. There was cDNA produced in the capping and cDNA strand synthesis so I am going to try and optimize the PCR reaction some more. After speaking with Jessica, we decided to go ahead and order the Platinum Pfx DNA polymerase that the kit calls for and also run a positive control simultaneously with the generacer reaction.
I am still trying to get the reactions to work and have a few new things that I will be working on this week and next. I will let you know how it all goes and if I have any more success.

Week of October 3rd to October 9th

This week I recieved my new glyma02g02430.1 3' primer. I ran PCR with this new primer and a clear band was visible. I extracted this band from the gel for use in the TOPO cloning. I recieved the remaining supplies necessary for TOPO cloning and will work on this in the upcoming week. I performed the 5' oligo capping via the GeneRacer kit again. I will use the newly prepared cDNA to run PCR and hopefully obtain bands for the 5' UTR regions, which can be used in TOPO cloning. Hopefully, the second run with the generacer kit was more successful.

Week of September 26th through October 2nd

This week I wasn't able to proceed much with the experiment. I had to order some supplies in order to clone the 3' DNA segments and have yet to receive them. In order to proceed with the 3' region of glyma02g02430.1 I designed a new primer for the 3' end and as soon as that comes in I will go ahead and use that in PCR to try and amplify that region for cloning as well. Hopefully this week I will be able to perform the 5' oligo capping and RACE in order to amplify these sequences more successfully.

First Month of Work

Over the summer, I spent ample time preparing for the laboratory work necessary for this project. I researched the RACE     protocol, which is the rapid amplification of cDNA ends. I began to understand how the protocol works and the necessary steps that must be taken. This process begins with RNA which is extracted from fresh or frozen tissue. Then, a specific oligo is added to the 5' end in place of the cap structure to allow easy identification of the region between the RNA cap and the start of the coding region. The RNA is reverse transcribed into cDNA using a reverse transcriptase. Then, the specific DNA sequences of interest can be amplified using a gene specific primer and a primer specific to the oligo added or an oligo dT primer for specific for 3' poly A tail.  With these amplified regions, the DNA can be sequenced directly or cloned first and then sequenced. This will provide information on the 5' and 3' untranslated sequences that are upstream and downstream to the genes of interested. The three genes I am studying are from the Glycine max genome. The identifiers are glyma01g05050.1, glyma02g02430.1, and glyma18g16170.1. These genes have yet to be characterized and that is the bulk of what I have been working on to date. They are classified as WRKY genes, which have been studied in Arabidopsis thaliana and appear to be involved in defense mechanisms against disease and illness. 

My work this semester began with RNA extraction from root tissue of an 18-day old w82 glycine max plant and root tips of a 3-day old w82 glycine max seedling. This extraction was performed with a Qiagen RNeasy Plant Mini Lit and followed by a DNA free treatment to purify the RNA obtained and remove any DNA or DNase contamination.  Next, I utilized the Invitrogen GeneRacer Kit to perform the RACE protocol using only the RNA from the root tissue to start. This kit included all the necessary reagents to oligo cap the RNA and reverse transcribe it into cDNA. In addition, I designed 5' and 3' gene specific primers for each WRKY gene of interest based on the DNA sequence. With these gene specific primers and primers specific for the oligo and 3' poly A tail, I attempted to amplify the upstream and downstream regions of these genes. This was not as successful as originally hoped and much optimization was needed in the PCR reaction. I had to adjust the annealing temperature and dilute the DNA to a more manageable concentration in order to successfully amplify these regions. Ultimately, I was only able to obtain the 3' downstream regions of the glyma01g05050.1 and glyma18g16170.1 genes, which was verified by performing gel electrophoresis with a slow melt 1% agarose gel. The slow melt gel was used because it allowed extraction of the gel bands to be used in TOPO cloning. This is the step that I am currently at and will be working on next. By cloning these sequences into the TOPO vector, I will then be able to sequence them. 

I was able to determine that the 5' capping was not successful with the use of two different positive control genes, Actin 1 and Beta Tubulin 1. Therefore, I must attempt again to perform this procedure with the GeneRacer kit. This will be performed within the next week or two. Hopefully, this second attempt is more successful and will allow the amplification and identification of the 5' upstream regions. As for the glyma02g02430.1 gene I will design new primers at different locations. I hope this will allow the PCR amplification to be more successful.