Rotation Notebook: Blackburn Lab, August-October 1983

This is a notebook within a series on Greider's experimental work at the Blackburn lab before rotating to other labs. The notebook detailed methods to cut plasmids with a specific enzyme in order to check the contents after complete digestion. Greider then ran a gel on cut plasmid fragment and set up a to-do list for the week. The list began with cutting DNA with a specific enzyme to removing it with glass beads and running a transformation test on the DNA. In Greider’s second lab rotation, she was cloning telomeres in yeast vectors. The Yeast vectors were a different organism with a different telomeric structure than Tetrahymena. Greider was using telomeres of Trypanosoma brucei to stabilize the linear plasmid in yeast However, T. brucei had caused some problems as the organism contained long barren regions where no restriction enzyme would have been able to cut. It is between a telomere linked segment and the end of the chromosome. The region would eventually cause difficulties in cloning. Decided to use T. cruzei instead of the species brucei because when cutting with Bal 31 it creates a more manageable size than T. brucei. Greider created a broad outine of project in rotation. Began to grow yeast cultures and purify their linear plasmids. Greider then grew cells in media, inoculate and harvest cells. After harvesting she created spheroplasts, which removed the cell wall and isolated the short linear plasmid. The plasmid was cut into 12 kilobase bands and ran it through agarose gel. Greider then cut the linear plasmid on both ends, for both Trypanosoma and yeast. Then Greider ran a transformation study on those ends. Greider seemed to be interested in those that didn’t fully transform after several days of growth in the medium. Found some new information, the A2C4 strain that she is trying to transform into a circular. However, Greider did not know the real genotype of this strain A2C4. Then Greider used the circular strain from Stillman to do transformation studies. This strain should grow faster than the A2 because it can take up average from the media. Then Greider cut the linear Plasmid DNA from an early Leptomonas DNA with restriction enzyme and ran a mini gel and check for complete digestion. However, the Plasmid didn’t cut because there was not much there. Greider used colonies from her 11/10/83 Transformation study which produced 536 colonies. The cells are growing on the surface rather than in the medium. Some from the carrier group of colonies and others from other transformation. Greider streaked four section on one plate of each- found that the cells are growing slowly. However, after analyzing the result Greider thought it was possibly something wrong with the media. Greider extracted the DNA from the available through glass beads spun down the DNA into a pellet. Greider ran a gel, however the result was too hot at first but didn’t cover with buffer until DNA well into gel. There was group meeting with the Blackburn lab. Greider informed the lab that she was working on the Telomere book, which was published in 1995, as well as a meeting with Blackburn.

Greider moved into the Ariel Lab and had her first meeting. Greider told the lab on her work regarding the Purification of the Human Telomere and RNA Identification. She essentially was looking for an RNA that co purifies with activity and assay for activity across the columns. She labelled the 3 prime and 5 prime DNA and detailed list of Human Telomerese Purification. She included a matrix, purification technique, and she commented on the process and results.

Greider moved into Jennifer’s lab and outlined the purpose for her work to the lab. Greider detailed methods and gave examples of dilution and experiments. Greider included quantitative analysis and summarized her work. In a more detailed presentation for Jennifer’s lab where Greider diagrammed clones with repeats and divergent repeats. Greider also compared areas that were rich in Adenosine and Thymadine.

In Thomas’ Rotation, Greider was rearranging pieces of DNA. The experiment rearranged different karyonides in macronucleus. Greider found three different repeat classes from macro nuclear DNA that were found within the initial clone. The base pair sequence was cloned, repeated, and sequenced. The notebook concluded with a synthetic DNA probe by analyzing the total RNA and found a single band.