Laboratory Notebook 3: In Vitro 26-55/FR 1-12, 1985 - 1986

Began with a summary of experiments that she had done so far. IV 27- mated cells to set them up and lysed and spun the cells. Mixed the cells to create a pellet of nucleate. Began testing different extracts for the nucleate and set up reactants (G4T2 and NTP). Ran the product through the gel and the bands that were produced became fuzzy. In 28 and 29 tested both hot and cold rNTP’s and fit them into tubes. Ran the tubes through a titration.

IV 30- rNTP is not well incorporated into the base pair pattern of nucleotides. Tried to figure out what the rNTP is doing in the process so she ran it through a gel

IV 31- loads a gel, uses specific extracts, reactions, nucleotides. After the experiment, she tries to determine how much excess DNA she is adding. Reviews different preparations to extract DNA, such as the measurements of buffer solutions, reactants, cell mating processes, and suspension.

IV 32-Uses yeast DNA (G4T4)from IV 27 spun it and the resulting reaction created a big white pellet. She then loadedthe gels with white and blue markers. IV 33- uses Trypanasoma extract and introduces it into Urea gel. Extracted the DNA from the gel and spun it. Concentrated it to clean the nucleotides. After running the subsequent gel, she decided to count the number of oligonucleotides. Afterwards, she tested the filter system of the gel

Finds that the agarose beads seem to absorb the DNA so that the result is unclear in the amount of DNA in each band.

Sets up another incorporation assay, in which she did the reaction conditions similar to previous experiment.

Group Meeting: Discusses with the group about adding telomere nucleotides and hot nucleate, and then run the gel. Discussion of which oligonucleotides worked in which experiments. A lot of procedures/ experiments involved sucrose.

Salt extraction had created problems. Tink it might be possible that the extract was done the “old way”.

Conclusion: Purify DNA more

The rest of the discussion was Greider gathering pertinent information regarding purification and different processes.

IV 34- Diluting the extract and treated the extract in tubes. Created a buffer and combined it with reactants. Sent it through a gel,autoradiograph of the image.

IV 35-Compared the use of paramecium cells with Tetrahymena cells and extracts. Testing several extract with Trypanisome with that of Paramecium and Tetrahymena. Loaded a gel of all three.

IV 38- The notes are based on group discussion. Conducts tests on some of the salts in the experiments. Adding them to extracts and loads the gel. Based on the results of the gel, she does some calculations to figure out conductivity of the gel. Also does a time course incorporation like she did in other experiments before.

IV 40- She varied the amount of salt in extract experiments and graphs the results, along with creating a gel.

IV 41- She conducted yeast extract experiments. Made spheroplasts, or a cell without the cell wall and uses an enzyme to degrade the celll wall. Then she collected and suspended the cel in a solution. Set up an assay and a gel along with the suspension.

006- Added salt to a mated cell extract. Conducted several gel studies, with assays using different enzymes than before. drew graphs throughout the folder, mostly pertaining to gel studies. However, she was having problems with some of her experiments but was able to correct it. She found that it was a problem with reaction buffer.

Also of note: correspondence between Greider and Elizabeth(1986). Greider was detailing what went wrong with the experiments. She was also trying to get a post doc position in other labs. At the same time trying to extract RNA by running a crude extract. She seemed to have been able to secure a talk on the 30th of September.

She continued with her mated cell extract experiments. Spun down the cells and reduced them down to a pellet. Then poured the protein gels for the pellet. While the pellet DNA was running she reviewed the experiments she had run so far and remarked on each one.

Did three separate experiments to fraction and isolate RNA. Created a buffer to ligase RNA and runs it through gel and seemed to be rather successful in doing so.

In other experiments where she is fractioning the DNA, the DNA are dying in the extracts. Tried another series of experiments fractioning RNA segments. She came to a realization, that what she really wanted to know was how the enzyme (Telomerase) worked and how it knew to elongate itself with G4T2. Began with testing MNase activity, which destroyed both DNA/RNA double and single strand. Found the general idea of her work was labelling the RNA’s and to see if she could identify a RNA that co-purifies with the DNA. So she repeated several RNA experiments to isolate/label/purify it. Afterward ran several gels to do so.

Also decided that instead of using Tetrahymena cells,she would use Euplates cells from a different researcher. The Euplate cells offered no elongation, similar to yeast.