Tinsley spent her summer working with Dr. Jay Kormish from the University of Manitoba in developmental biology - hoping to better understand the genetic basis for how cells coordinate changes during organ development, using nematode worms (C. elegans) as a model
Learning procedures and protocols and improving my molecular biology techniques
Worm Picking:C. elegans worms are kept at 15°C and live on agar plates covered with E. coli to feed on. About twice a week, each plate is picked over and the adult hermaphroditic worms are transferred to a fresh plate, to prevent the worms from starving out and to continue on the various mutant strains.
Primers:C. elegans contains 6 chromosomes, 5 autosomal and 1 X chromosome. The primers are specific sequences found within its genome that are used for PCR amplification of the worm’s DNA. We have to use some math skills to figure out how much of each primer is
Continuing to improve my molecular biology techniques by carrying out experiments of C. elegans DNA and becoming familiarized with common protocols in the Kormish Lab.
PCR prep: More math skills and careful pipetting are required to prepare master mixes for PCR. Lysed C. elegans DNA is added to each tube; with each tube containing specific sequences for each chromosome. PCR will create lots of copies of the DNA. It is crucial to keep this step as clean and sterile as possible so that there is no contaminant DNA added to the PCR (which could later show up on the gel). After this step, the DNA will be cut up with an enzyme and imaged using gel electrophoresis.
My worm box,
containing three strains of C. elegans worms. Twice a week, 8-10 worms are transferred to a new plate of fresh E. coli for the worms to feed on. Old plates are kept as a precaution. If one of the strains begins to die off, we can go back to older strains to see where things went wrong and use these older worms to keep the strain alive.
After PCR and the DNA has been cut with an enzyme, everything is added to an agarose gel through which an electric current is run. DNA is negatively charged, so it is pulled toward the positive end. We added a dye to be able to see the DNA progress. After 2 hours, we remove the gel and take a picture using UV light, giving the image on the right. Each band shows DNA of different lengths. A process of several days of careful pipetting comes down to one picture, and it is only then that we can see how well the experiment was performed.
Scanning through thousands upon thousands of A, T, G, C sequences to find the perfect combination to be used in a PCR. Learning how to use various programs such as WormBase, BLAST and Primer3 to help with this design process.
Gel Electrophoresis to visualize quantity of DNA from PCR for DNA collection to send off for sequencing.
Setting up a cross between wild type male C. elegans and hermaphroditic C. elegans to confirm the inheritance of mutant alleles in pharynx gland cell development.
Analyzing the preliminary results of the cross mentioned above. Using the fluorescence microscope to visualized yellow fluorescent protein that was added to the pharyngeal cells of the C. elegans to determine whether the pharynx gland cells underwent normal development or the mutant allele was passed on to this generation.
Purifying DNA samples from PCR of different C. elegans mutants to send off for Sanger sequencing. This will confirm the genes involved in the mutant phenotypes.
Using the Nanodrop machine to evaluate the purity of DNA samples collected above.
Using liquid nitrogen to extract RNA from worm eggs. This is to create cDNA (just the part of the DNA with the gene that creates a worm phenotype), so that we can confirm the gene that is causing the mutation.
The primers I designed were ordered. I had to prepare stocks that we could use and test them all to make sure they work as expected at the correct temperature.
The past two weeks have been fixing up the primers by testing them and planning a chromosome mapping experiment (below). I am currently focusing on markers found on chromosome 1 and in the coming two weeks will be collecting data from all of these markers in the mutants worms to determine which gene is most likely causing the mutation.
I have completed the chromosome mapping experiments and compiled all the results in a table to get an idea of what marker the unknown gene is closest to. Comparing this to a list of candidate genes will tell us which gene most likely has the mutation. I have narrowed it down to two genes from a list of seven and I am currently researching papers that involve these genes in pharynx development.
This will give an idea of which versions (alleles) of the gene could be affected by the mutation. Once we can determine which allele is most affected, we will have a better idea of the overall pathway of the g1p cell development. I am also going to begin the same mapping experiment on chromosome X, to see what other genes are involved.