Laboratory Notebook 6: In Vitro 87-91/FR 70-95/RNA 29-65, 1986 - 1987

Display Title

Laboratory Notebook 6: In Vitro 87-88/FR 70-93/RNA 33-47, 1986 - 1987

Source

box: 03 folder: 03-04 (text)

Extent

2 Folders

Description

The notebook begins with a series of fractionalization and kinased gel studies using an RNA strand. Greider then ran the sequence through a gel and also ran subsequent gels through Urea and Acryl gels. Greider then isolated the sequences from her series of gels and fractionalizations. The isolated RNA was spun, the suspension was poured out and dried, in order to extract RNA. Grieder then ran the extract through a gel and exposed the gel for an hour. After the experiment, Greider drew a graph of fractionalization of starved cells, using Tetrahymena cells.

After drawing the graph, Greider ran an RNA sequencing experiment which diluted the buffer she had created using an enzyme. In total Greider used five enzymes over all for each dilution. Greider also used another organism, outside of Tetrahymena. She used Paramecium and in the same vein as her Tetrahymena she starved cell extracts. Then Greider took the cells, spun them, and then packed the cells into a pellet. Afterwards, Greider ran two different preparation gels using a five prime kinase enzyme in one and three prime pCP enzyme on a single strand of RNA. An interesting note, a five prime sequence was a prominent band, Greider noted it as a mystery RNA. Greider noted the mystery band as band number 298. She then sequenced the band in other RNA tests and ran a gel on the band. In order to find the mystery band, Greider ran a 300 volt gel to locate the band in relation to its markers. Greider she also found that certain sets of sequences were in the wrong direction. It could also be that the nucleotide was wrong because a break in the gel. Greider noted to check gel sequences for certain bases, change nucleotides to cytosine and Urasil. The Greider ran a series of fractionalization experiments, alongside oligonuceoltide counts to determine if shifts occurred in the oligonucleotides. Then Grieder ran the nucleotides through an RNAse enzyme, wanting to determine if it was the cause of the decreased activity in the oligonucleotides. Finally, Greider conducted a series of experiments with and without the RNAse enzyme. Another interesting note Greider also conducted experiments using mammalian cells from Stillman lab, which Greider washed, extracted its nuclei, and loaded it into a gel. Greider also led a group meeting to discuss the telomerase enzyme. She stated that the overall goal was to identify and characterize the RNA component of the telomere. The synthetic telomere that she used seemed to repeat from the beginning, using the sequence band G4T2, and noted that the band shifted from different three prime ends. Greider also gave a list for group meeting where she used the enzyme RNP and identified the RNA component using purification techniques. Greider then purified the telomere activity through certain columns in a gel to see which RNA component copurifies. Greider then sequenced the RNA component and noted that the last time she was trying to clone the gene for the telomerase, she was able to locate it at 159 kilobases on the sequence. As for the RNA sequence, the gene showed the sequence, specifically the C4A2 sequence, which could be a template for the gene. The notebook concluded with Greider discussing the evidence for the 159 RNA strand in the gene as well as her reconstitution experiments.

Scope and Contents

The notebook begins with a series of fractionalization and kinased gel studies using an RNA strand. Greider then ran the sequence through a gel and also ran subsequent gels through Urea and Acryl gels. Greider then isolated the sequences from her series of gels and fractionalizations. The isolated RNA was spun, the suspension was poured out and dried, in order to extract RNA. Grieder then ran the extract through a gel and exposed the gel for an hour. After the experiment, Greider drew a graph of fractionalization of starved cells, using Tetrahymena cells.

After drawing the graph, Greider ran an RNA sequencing experiment which diluted the buffer she had created using an enzyme. In total Greider used five enzymes over all for each dilution. Greider also used another organism, outside of Tetrahymena. She used Paramecium and in the same vein as her Tetrahymena she starved cell extracts. Then Greider took the cells, spun them, and then packed the cells into a pellet. Afterwards, Greider ran two different preparation gels using a five prime kinase enzyme in one and three prime pCP enzyme on a single strand of RNA. An interesting note, a five prime sequence was a prominent band, Greider noted it as a mystery RNA. Greider noted the mystery band as band number 298. She then sequenced the band in other RNA tests and ran a gel on the band. In order to find the mystery band, Greider ran a 300 volt gel to locate the band in relation to its markers. Greider she also found that certain sets of sequences were in the wrong direction. It could also be that the nucleotide was wrong because a break in the gel. Greider noted to check gel sequences for certain bases, change nucleotides to cytosine and Urasil. The Greider ran a series of fractionalization experiments, alongside oligonuceoltide counts to determine if shifts occurred in the oligonucleotides. Then Grieder ran the nucleotides through an RNAse enzyme, wanting to determine if it was the cause of the decreased activity in the oligonucleotides. Finally, Greider conducted a series of experiments with and without the RNAse enzyme. Another interesting note Greider also conducted experiments using mammalian cells from Stillman lab, which Greider washed, extracted its nuclei, and loaded it into a gel. Greider also led a group meeting to discuss the telomerase enzyme. She stated that the overall goal was to identify and characterize the RNA component of the telomere. The synthetic telomere that she used seemed to repeat from the beginning, using the sequence band G4T2, and noted that the band shifted from different three prime ends. Greider also gave a list for group meeting where she used the enzyme RNP and identified the RNA component using purification techniques. Greider then purified the telomere activity through certain columns in a gel to see which RNA component copurifies. Greider then sequenced the RNA component and noted that the last time she was trying to clone the gene for the telomerase, she was able to locate it at 159 kilobases on the sequence. As for the RNA sequence, the gene showed the sequence, specifically the C4A2 sequence, which could be a template for the gene. The notebook concluded with Greider discussing the evidence for the 159 RNA strand in the gene as well as her reconstitution experiments.

Preferred Citation

Laboratory Notebook 6: In Vitro 87-88/FR 70-93/RNA 33-47, 1986 - 1987. Carol Greider Collection, Cold Spring Harbor Laboratory Archives Digital Repository. 88-1525500. Update 2025-03-18.

Credit Line

Courtesy of Cold Spring Harbor Laboratory Archives.

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Category

Lab Notebooks

Publications

Level

file

Collection

Carol Greider Collection

Resource ID

CWG

Component Unique Identifier

CWG_01_011

Reference ID

b93d3d6752156d32bb7c41918db1d9d5

Finding Aid Link

Laboratory Notebook 6: In Vitro 87-91/FR 70-95/RNA 29-65