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.