Laboratory Notebook 1: In Vitro 1-8, 1984 - 1985

Page 1

Greider's To Do List. Items on the list include creating telomere representative models, protein purification, enzyme kinetics and inhibition, macronuclear development, and lastly a research summary.

Page 2

Greider’s ideas for potential assays using exonuclease or endonuclease single stranded DNA in extracts. One assay includes using ExoIII, a substrate, and cordycipen (a derivative of the nucleoside adenosine). She mentions decreasing the concentration of labeled DNA, adding various amounts of cold DNA, using pBR 322 (a plasmid vector), and then running a sequencing gel. Another assay she proposes is using end labeled single-stranded DNA, taking the above fragment and the possibility of denaturing or not denaturing it.

Page 3

Greider draws double stranded DNA, the plasmid pMC, and she also writes down mnI\Bst, which are restriction enzymes.

Page 4

Drawings of what TdT (terminal deoxynucleotidyl transferase), λ Exonuclease and Exonuclease III treated DNA would look like in combination with cordycipen and kinase. TdT adds nucleotides to the 3’end of a DNA strand and does not need a template. Exonuclease III digests nucleotides starting from the 3’ end and λ Exonuclease digests nucleotides starting from the 5’ end.

Page 5

A to do list including items such as looking at reaction concentrations for each experiment, rDNA labeling, Hha P11b fragments where G+C (guanine and cytosine nucleotides) are constant, scanning IV2, and ammonium sulfate precipitation. She also writes down the name Schachman, which might correspond to former UC Berkeley professor, Howard Schachman.

Page 6

To do list continued. Includes doing a polym (polymerase) time course and to filter the assay. Also concludes that IV3 (In-Vitro Experiment 3) was blunt.

Page 7

Greider details the enzymatic properties of a reaction. She includes the time course of a native vs a heat denatured extract. The heat denatured extract showed no reaction occurring, demonstrating that this reaction is susceptible to heat, which is expected for an enzyme-catalyzed reaction. This page also includes the initial rates measurement using dATP vs dTTP, if the oligomer concentration dependence is linked to energy dependence are detailed. She includes using a gel and TCA (trichloroacetic acid) precipitation assays, which is used to concentrate proteins or get rid of contaminants prior to gels. She also mentions n-ethylmaleimide and iodoacetamide, which are inhibitors of cysteine peptidases, and the reaction dependence to them. Greider suggests that cysteine might be in the active site of the enzyme, that we now know as telomerase, which would explain why the reaction is sensitive to n-ethylmaleimide and iodoacetamide, since they modify the cysteine residue in proteins. She also mentions end-labeled oligos and HPLC and rATP (ribonucleoside adenosine triphosphate) generating systems, where she notes she should look at in-vitro splicing and extract the experiments from Sharp et al.

Page 8

Greider describes translocation for Tetrahymena and yeast. She also mentions what the active site of telomerase includes, which comprises of a recognition region, a dGMP acceptor site, a dGTP binding site, a dTTP binding site. She notes that for trypanomosomes it also includes a dATP binding site or it can share the dGTP binding site because it can bind ATP.

Page 9

Greider depicts the general structure of telomere terminal transferase, also known as telomerase. It shows how the enzyme works by elongating and translocating DNA to add telomere DNA sequences.

Page 10

The end of Greider’s depiction of the mechanism of telomerase.

Page 11

Greider notes that she needs to add oligos, dGMP (deoxyguanosine monophosphate), dGTP (deoxyguanoxine triphosphate), and dTTP (deoxythymidine triphosphate) to see whether telomere sequences would be added to those oligos. This would be done by using gel assays. She also mentions doing oligos curves for the different oligomers she will use by doing TCA precipitation assays.

Page 12

DNA Oligomer Request Form added to Greider’s lab notebook from Carol Greider dated April 17, 1985. She requested a yeast telomere sequence. It notes that she is in the Blackburn Lab in Stanley Hall.

Page 13

DNA Oligomer Request Form added to Greider’s lab notebook dated April 17, 1985 from Carol Greider and Eric Henderson, a postdoctoral fellow in the Blackburn Lab. The sequence requested was a short telomere sequence, identical to the one requested on January 30, 1984.

Page 14

DNA Oligomer Request Form added to Greider’s lab notebook dated January 30, 1984 from Carol Greider and Eric Henderson. The sequence requested is the Tetrahymena telomere sequence. It is not the sequence that telomerase adds nucleotides to, it is the corresponding strand’s sequence. Later, at an unspecified date, Greider wrote "ordered + received".

Page 15

DNA Oligomer Request Form added to Greider’s lab notebook dated January 30, 1984 from Carol Greider and Eric Henderson. The sequence requested was the Tetrahymena telomere sequence.

Page 16

DNA Oligomer Request Form added to Greider’s lab notebook dated January 30, 1984 from Carol Greider and Eric Henderson. The sequence requested was a short telomere sequence, identical to the one requested on April 17, 1985.

Page 18

March 7, 1984. This page was inserted into Greider’s lab notebook. It shows the sequence of pCA2, a plasmid. Greider notes the 5' and 3' ends. It also shows where the restriction sites are located and the corresponding restriction enzymes.

Page 19

This page was inserted into Greider’s lab notebook and it is dated November 24, 1984. It shows the DNA sequence of DNA 11b.1tr.

Page 20

November 24, 1984. The second half of the DNA sequence of DNA 11b.1tr.

Page 21

This page was inserted into Greider’s lab notebook and it is dated November 24, 1984. It shows the DNA sequence of DNA 1737.1tr.

Page 22

November 24, 1984. Second half of the DNA sequence of DNA 1737.1tr. Greider has circled U (uracil), a nucleotide that shows up in RNA sequences.

Page 23

November 24, 1984. The DNA sequence of DNA 1737.1tr. Greider again has circled U (uracil) and she has also written a question mark next to it.

Page 24

July 12, 1984. Location of the restriction sites of the rDNA Palindrome of Tetrahymena and the corresponding restriction enzymes.

Page 25

July 12, 1984. Location of the restriction sites of the rDNA Palindrome of Tetrahymena , the corresponding restriction enzymes and the fragment lengths the restriction enzymes would produce.

Page 26

July 12, 1984. Location of the restriction sites of the rDNA Palindrome of Tetrahymena , the corresponding restriction enzymes and the fragment lengths the restriction enzymes would produce. Greider underlines the 1535 location of the HhaI restriction site and its fragment lengths and ends.

Page 27

July 12, 1984. Location of the restriction sites of the rDNA Palindrome of Tetrahymena, the corresponding restriction enzymes and the fragment lengths the restriction enzymes would produce.

Page 28

July 12, 1984. Location of the restriction sites of the rDNA Palindrome of Tetrahymena, the corresponding restriction enzymes and the fragment lengths the restriction enzymes would produce.

Page 29

July 12, 1984. Location of the restriction sites of the rDNA Palindrome of Tetrahymena, the corresponding restriction enzymes and the fragment lengths the restriction enzymes would produce.

Page 30

July 12, 1984. Location of the restriction sites of the rDNA Palindrome of Tetrahymena, the corresponding restriction enzymes and the fragment lengths the restriction enzymes would produce.

Page 31

July 12, 1984. Location of the restriction sites of the rDNA Palindrome of Tetrahymena, their corresponding restriction enzymes and the fragment lengths the restriction enzymes would produce and Commercially Available Restriction Enzyme Library Map of the rDNA Palindrome of Tetrahymena.

Page 32

July 12, 1984. Continuation of the Commercially Available Restriction Enzyme Library map of the rDNA Palindrome of Tetrahymena. At the bottom, the restriction enzymes that do not have a restriction site in the rDNA Palindrome of Tetrahymena are listed.

Page 33

July 12, 1984. DNA sequence of the rDNA Palindrome of Tetrahymena containing where various restriction enzymes have their restriction site.

Page 34

July 12, 1984. Continuation of the DNA sequence of the rDNA Palindrome of Tetrahymena and where various restriction enzymes have their restriction site. Greider draws an arrow underneath the restriction enzyme, HhaI, noting her interest in it.

Page 35

July 12, 1984. Continuation of the DNA sequence of the rDNA Palindrome of Tetrahymena and where various restriction enzymes have their restriction site. Greider noted her interest in the restriction enzyme, BstXI, and its restriction site by circling it.

Page 36

July 12, 1984. Continuation of the DNA sequence of the rDNA Palindrome of Tetrahymena and where various restriction enzymes have their restriction site. Greider draws an arrow on top of the restriction enzyme, HhaI, which corresponds to the arrow she drew on a previous page

Page 37

July 12, 1984. Continuation of the DNA sequence of the rDNA Palindrome of Tetrahymena and where various restriction enzymes have their restriction site.

Page 38

July 12, 1984. Continuation of the DNA sequence of the rDNA Palindrome of Tetrahymena and where various restriction enzymes have their restriction site.

Page 39

July 12, 1984. Continuation of the DNA sequence of the rDNA Palindrome of Tetrahymena and where various restriction enzymes have their restriction site.

Page 40

July 12, 1984. Continuation of the DNA sequence of the rDNA Palindrome of Tetrahymena and where various restriction enzymes have their restriction site.

Page 41

July 12, 1984. Continuation of the DNA sequence of the rDNA Palindrome of Tetrahymena and where various restriction enzymes have their restriction site.

Page 42

July 12, 1984. The Commercially Available Restriction Enzyme Library Listing. Greider shows special interest in the restriction enzyme BbvI and the sequence surrounding its restriction site. She also underlines the restriction site of ThaI.

Page 43

This page is inserted into Greider’s lab notebook (dated 1984-1985). It describes how there are two kinds of nuclei (macronuclei and micronuclei) present in ciliated protozoans, such as Tetrahymena. Macronuclei are formed from micronuclei. Macronuclear DNA molecules are linear, subchromosomal, and autonomously replicating pieces (ARPs) and contain telomere sequences at both ends (C4A4 or C4A2 depending on the species). Greider notes that the precursor micronuclear DNA molecules are missing these telomeres, so they must have been added or transported by some process in order for them to be present in the macronuclear DNA. She sets out to try to elucidate the “specificity of this process” by analyzing a gene that is associated with the ARPs of Tetrahymena. She chooses to analyze the ribosomal RNA genes because they are the “shortest, most abundant, and most readily purified molecules”. She notes that the ribosomal RNA genes of Tetrahymena have a nonpalindromic center that separates the two palindromic halves of the ribosomal RNA gene. This page ends in the middle of a sentence contrasting the amplified macronuclear DNA.

Page 46

This page is inserted into Greider’s lab notebook (dated 1984-1985). It depicts the EcoRI (a restriction enzyme) insert of plasmid vector, pTt503. This would be where a fragment would be inserted if the plasmid and the fragment were treated with EcoRI.

Page 47

This page is inserted into Greider’s lab notebook (dated 1984-1985). It depicts the EcoRI (a restriction enzyme) insert of plasmid vector, pTt503. This would be where a fragment would be inserted if the plasmid and the fragment were treated with EcoRI. This is the second copy of this document in this notebook.

Page 48

The bottom half of this page is dated May 23, 1983. The upper half doesn’t have a date, but it is linked to the notes of the bottom half of the page, so it should be around May 23, 1983. Clones of T. brucei (Trypanasoma brucei) DNA were made and were probed with pTbr1, which probes for the small or large subunit of rRNAs, or Gx + Tx + As probes. Out of 28 clones, she chose 4 clones to do a detailed restriction mapping using 5 different restriction enzymes.

Page 49

May 23, 1983. A picture of the gel (21 slot/well 1.2% agarose gel) is shown on the bottom half of the page. The gel was used to run the Trypanasoma brucei clone DNA fragments produced by the restriction enzymes that she listed on May 23, 1983. A detailed diagram of what each slot/well contains in the gel is also included.

Page 50

Picture of the gel is dated around May 23, 1983 and was exposed for 3 days and probed with G+T+A. Sample numbers on top of gel correspond to sample numbers located here

Page 51

List of things Greider wants to consider for future in vitro experiments. This includes concentrating PEG, reaction conditions including ionic strength and finding out if rNTP’s are inhibiting the reaction.

Page 52

This page is inserted into Greider’s lab notebook (dated 1984-1985). It depicts the EcoRI (a restriction enzyme) insert of plasmid vector, pTt503. This would be where a fragment would be inserted if the plasmid and the fragment were treated with EcoRI. This is the third copy of this document in this notebook.

Page 53

This page is inserted into Greider’s lab notebook (dated 1984-1985). It depicts the EcoRI (a restriction enzyme) insert of plasmid vector, pTt503. This would be where a fragment would be inserted if the plasmid and the fragment were treated with EcoRI. This is the fourth copy of this document in this notebook; however, on this copy Greider has added the mnI insert.

Page 56

Greider references 2 articles and where they can be found in the journal, Nature. The full titles for the articles are “Vaccine development: Hybrid vaccinia virus for mass hepatitis immunization?” and “Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes”.

Page 57

March 23, 1984. Greider details an experiment probably from a talk by someone named Jim. It is not specified who Jim is, although there was a man named Jim Bliska that did his first graduate school lab rotation in Elizabeth Blackburn’s lab, so perhaps this is the Jim she is referencing.

Page 64

Start of In Vitro Experiment 1.

Page 65

April 20, 1984. Greider details an experiment that involves Greider cutting pIIb with the restriction enzyme, Hha in order to isolate 1650 bp (basepair) and 1400 bp fragments with C4A2 (telomere DNA sequence). She mentions calibrating Slow Bal 31, which is an exonuclease that degrades DNA at the 3’ and 5’ ends. “Slow” refers to one of two forms that Bal 31 can have (Slow vs. Fast). The two forms are kinetically and molecularly different. Greider cuts another plasmid vector, pBR 322, with the restriction enzyme, RsaI. She expects to see 3 different-sized fragments (2117 bp, 1565 bp, and 680 bp). She mentions adding Bal 31 and expecting to see the 680 bp fragment “disappear”. She also details why the pBR digest is good.

Page 66

April 21, 1984. Greider mentions that the April 21, 1984 gel can be found in the "Telomere Cloning" Notebook. She estimates that the total concentration of DNA of the plasmid, pBR 322 is 4.2 µg in 100 µL. In the second half of the page, Greider calculates the desired concentration of the exonuclease Bal 31 to be used in the experiment. Greider also makes a different calculation, using a different colored pen, and notes that her previous calculation was wrong. The use of a blue ink pen vs a black ink pen that she was using throughout the page indicates that the blue ink calculations were added after April 21, 1984.

Page 67

April 21, 1984. Greider details the composition of the buffer that the exonuclease Bal 31 was diluted in, although Greider notes there is a discrepancy between what the spec sheet says and what the enzyme concentration is, so she decides to not dilute the Bal 31. She notes using 25 µg and 1 µL of Bal 31 and using time points: 5, 10, 20, 30, and 60 min.

Page 68

This page continues the experiment from April 21, 1984. Greider notes how she set up the Bal 31 digest. This page also contains the prep gel (1.5% agarose run at 50V for ~12 hours) for the Hha restriction enzyme digest of pIIb . Greider notes that the running time was too long probably because the bands are running off of the gel.

Page 69

April 22, 1984. Greider details cutting pIIb again with the restriction enzyme, Hha. Greider notes that the gel from that experiment is in the “Telomere Cloning” notebook and that there was only a partial digest, so she decides to “add 2 µL of enzyme and incubate at 37° again”. This page also includes a mini gel (1% agarose run at 80V) of the Slow Bal 31 calibration at the time points described in the notes from the page dated April 21, 1984. Greider writes that she should have run a 2% agarose gel instead, maybe so that the bands in the ladder would be more clear and distinct.

Page 70

April 22, 1984. Greider concludes that the Bal 31 is slower than expected because the concentration of DNA was not ideal. This page also contains notes from the date April 24, 1984, that concludes that the pIIb Hha digest from April 23, 1984 is complete. Lastly the page contains a gel (1% agarose run at 40V for 14 hours) from April 28, 1984 which shows the pIIb Hha digest fragments. Greider notes that she will cut out the 2 top bands together and cut the 1400 bp separately.

Page 71

Greider makes calculations for the fragment sizes of a pBR 322 digest, but then she adds a note after the fact that says “WRONG” to indicate her original calculations had been wrong and she directs herself/readers to look at an entry that is dated July 19, 1984. On the bottom half of the page, she details planning an experiment which comprises of pooling the 3 fragments of the restriction enzyme digest of the plasmid pBR 322 together, adding the exonuclease Bal 31 and then splitting the resulting sample into three different reactions containing three different nucleases(one with ExoIII added, another with S1 added, and the third with λExo added). Then she plans to cut the resulting DNA product with Xba, a restriction enzyme, and finally run the DNA on a gel.

Page 72

Greider writes that she wants to calibrate the exonucleases, λExo and ExoIII.

Page 73

On April 30, 1984, Greider mentions calibrating the exonucleases, ExoIII and λExo and using a plasmid that has been linearized, pBR322, by the restriction enzyme Bam from April 22, 1984. She also mentions the enzyme, T4 DNA Polymerase, which she will also calibrate, and making buffers and she writes the components of buffers for ExoIII.

Page 74

April 30, 1984. Greider details the components of the λExo buffer and the T4 DNA Polymerase buffer and the calculations needed to set the components to their desired concentrations.

Page 75

May 1, 1984. Greider details how much ExoIII and λExo Greider needs to use to get 40 nucleotides/end/30 min.

Page 76

May 1, 1984. Greider details how much T4 polymerase to use to get 2 bases/min/end. Greider also details she needs new T4 polymerase buffer conditions for this and she writes her calculations on how to get those new buffer conditions.

Page 77

May 1, 1984. Greider details setting up 3 reactions for the enzymes ExoIII, λExo, and T4 DNA polymerase and what the components of each reaction are, the different time points for the reactions, and that each reaction will be run at 37°C. The DNA being used is pBR 322 linearized by the restriction enzyme Bam. She had also written down she was going to also run the T4 polymerase at room temperature, but then she scratched that part out. In order to stop the reaction at the desired time points, she writes that at each time point she will remove 50 μL of the reaction and add it to 5 μL of Bal 31 (exonuclease) stop buffer from April 21, 1984.

Page 78

May 1, 1984. How to dilute T4 polymerase ExoIII to give certain enzyme activities.

Page 79

May 1, 1984. Greider details what she did after stopping the three reactions detailed in https://cshl.smugmug.com/Carol-Greider-Archives/CWG-01-001/i-FFv2mqC/A. She details diluting the enzyme S1 from 95 U/ μL to 1 U/ μL. U is a unit of the enzyme’s catalytic activity. She also mentions doing ethanol (EtOH) precipitation.

Page 80

May 3, 1984. Greider mentions at the top that she “lost all the DNA somewhere” from her previous experiments dated May 1, 1984. She writes that she will try the reactions again using twice the amount of DNA and enzyme that she had used in her previous experiments, probably to increase the chances that the DNA won’t be lost again, and she details the components of each reaction. She also notes that the DNA used in the reactions is pBR 322 plasmid linearized with the restriction enzyme Bam (the same as in her previous reactions). She details at the end of the page that she will stop the reactions in 2 μL of 200 mM EDTA; however on May 4, 1984 (the day after) she scratches that part out and writes that she actually stopped the reaction in 5 μL of Bal 31 stop buffer. On May 4, 1984 she mentions doing a phenol extraction (Ø-OH extract) and ethanol precipitation (EtOH ppt) in order to purify and precipitate the DNA products of the reaction.

Page 81

May 4, 1984. Continuation of these experiments. She details bringing up the pellets that were produced by ethanol precipitation in a TE buffer and adding various reagents including the restriction enzyme EcoRII and BstNI buffer. Greider also writes that the EcoRII/Bam Digest of the plasmid pBR322 produces 7 fragments, two of which (682 and 245) she believes should be “Exo’d”, or used as a substrate for the exonucleases.

Page 82

May 4, 1984. A picture of the gel (2% agarose) of the ExoIII calibration is taped to the page. Greider notes that the gel shows that it looks all of the DNA got degraded. She proposes trying another reaction involving taking 2 μL of pBR 322 plasmid cut with RsaI and incubate. She also details doing four reactions. One reaction uses only SI buffer, another uses SI buffer + SI enzyme. The other two reactions use ExoIII buffer, with one reaction also have the enzyme ExoIII.

Page 83

May 6, 1984. Includes a picture of a gel (1% agarose run at 80V) that is taped to the page from the experiments from May 4, 1984. Bands are shown for every reaction except for the ExoIII + Buffer reaction. Greider speculates that the ExoIII enzyme is eating up all of the DNA and she suggests to try adding more DNA to the ExoIII reaction. In the bottom half of the page, she notes the reagents and the various concentrations of DNA that she used for the next reaction to maybe help solve the problem of all of the DNA being degraded from ExoIII. Greider also notes she will use a new sample of linearized plasmid pBR cut by Rsa. She attaches a picture of a gel (1% agarose run at 80V) that shows this new pBR cut by Rsa, which looks identical to the bands for the old pBR Rsa shown on top gel. This is expected since they should be the same DNA sequence.

Page 84

May 6, 1984. Greider notes that there was a phenol extraction after the Exo (ExoIII and λExo) digests. Ethanol precipitation was done next and the pellets that were produced by the precipitation was resuspended and then various components were added. The reaction was incubated for 1 hour. After, Greider notes that she did ethanol precipitation and brought up the resulting pellet. The resulting DNA products are shown in a picture of a gel (2% agarose) that is attached to the page. The gel shows the 16 reactions that were done using varying concentrations of DNA (4 reactions with ExoIII, 4 reactions with λExo and 4 reactions with T4 polymerase). Out of the 4 reactions done for ExoIII and λExo, only one for each showed bands. Perhaps this is because that reaction corresponded to the highest DNA concentration that was used. Greider did not write which bands correspond to which DNA concentration, but it makes sense that she would place the reactions from low concentration of DNA to high concentration of DNA used. Also the bands from T4 polymerase go from being faint to bright from left to right, which further suggests the order that Greider used to place the reactions in the gel. In general, the brightness of a band has a positive correlation with the concentration of DNA. Greider concludes that she wants to see sharper bands, so she should use a shorter time to run the gel.

Page 85

May 9, 1984. Greider writes that she will pool “the two GB extractions of pIIB Hha fragments” from May 3, 1984 and April 28, 1984 and run a mini gel to estimate the DNA concentration. Greider also mentions fine tuning her Exo reactions by using certain amounts of the linearized plasmid pBR 322 and also certain amounts of the ExoIII and λExo enzymes. It looks like she first wrote that she was going to use 18 μL for pBR Rsa, but then she wrote 16 μL on top of it. It also looks like she did the same for the amount of ExoIII and λExo enzymes. She first wrote 1 μL and then wrote on top of that 2 μL. The time points she uses are 2, 5, 10, 20 min. She also details how she set up the reactions for the ExoIII enzyme + ExoIII buffer reaction and for the ExoIII buffer only reaction. She also notes she did a phenol extraction and ethanol precipitation to purify and precipitate the DNA product and how she resuspended the resulting pellet.

Page 86

May 9, 1984. A picture of a gel (1% agarose run at 80V) is taped to the top of the page. The gel shows bands for 2 μL of phage T7 DNA cut with Kpn/PvuII and bands for 1 μL of pIIb cut with Hha along with some DNA concentrations. Greider also notes some calculations for the slow Bal 31 nuclease. Some are probably done after the notes at th top of the page since they are done with a different color pen. Also there are modifications to those calculations, also probably done later because the modifications are also in a different colored pen.

Page 87

May 12, 1984. A picture of a gel (2% agarose)is taped to with the results from the reactions detailed in May 9, 1984. It also includes the untreated pBR Rsa. Greider writes that she is a bit disappointed that the enzyme seems to be already digesting a lot of DNA at the 2 minute mark. She notes that she should dilute the Exo enzymes 1/10 and decrease the temperature of the reactions to 30°C in order to slow down the Exo digestions. From previous experiments, she was probably using 37°C as the reaction temperature.

Page 88

May 15, 1984. Greider makes calculations for the Slow Bal 31 nuclease that she had been using to stop the reactions, but she concludes that she would need to much Slow Bal 31 and she then makes the calculations for generic Bal 31.

Page 89

May 15, 1984. Greider suggests checking if the generic Bal 31 nuclease is working by checking the Bal 31 reaction at 10 and 20 min time points. But she then writes that she will do the enzyme calibration “right” and use 2, 5, 10, and 20 min time points using the pIIb cut by Hha (3 fragments). At the bottom of the page a picture of a gel (1% agarose run at 80V) is taped to the page. The gel shows that DNA is being degraded as time goes on, which is what Bal 31 should do.

Page 90

May 15, 1984. A picture of a gel (1% agarose run at 80V) is taped to the top of the page. The gel shows the results of treating pIIb Hha with Bal 31. Greider notes that the Bal 31 reaction worked but she is losing a lot of DNA in the phenol (Ø-OH) extraction and doesn’t have enough of “the prep digest to go on with”. In the bottom half of the page she details how to make more Bal 31 buffer.

Page 91

May 15, 1984. Greider notes doing the “large scale 4 min Bal 31’s at 28°C again”. A picture of a gel (1% Agarose run at 80V) is taped to the bottom of the page. Greider notes that the Bal 31 reaction worked and she has enough DNA to continue, in contrast to her previous experiment. She mentions using the DNA from the gel and pooling it together with DNA from yesterday’s reaction for use in Exo experiments.

Page 92

May 16, 1984. Greider writes 3 different reactions to do using S1 , ExoIII, and λExo, although she adds later to wait on the λExo reaction. The bottom half of the page is dated May 18, 1984. She notes that she wants to do one more ExoIII calibration at a low dilution. The time points used are 2, 5 and 4 min.

Page 93

May 16, 1984. Greider details resuspending the pellet in various reagents, incubating reaction the reaction and doing EtOh ppt (ethanol precipitation). A gel (1% agarose run at 70V) is taped to the page, showing the results of the experiment detailed on the previous page. Greider concludes from the gel that the exonuclease, λExo is faster at digesting DNA than ExoIII and she proposes using a more diluted λExo solution.

Page 94

May 19, 1984. Greider details how to prep more pIIb treated with the restriction enzyme Hha and the nuclease Bal31. A gel showing the results of that reaction and of untreated pIIb is taped to the bottom of the page.

Page 95

May 19, 1984. The components needed to make 1 mL of 10x and 2x Buffer for in vitro reactions plus their corresponding concentrations and volumes are detailed. Greider also notes adding DTT, rNTP’s, dATP, and dTTP to a 2x reaction mix.

Page 96

May 19, 1984. Details of how Greider set up her in vitro experiment using three different enzymes (S1, ExoIII, and λExo)for hot and cold reactions.

Page 97

May 19, 1984. A flow chart that includes inoculating Tetrahymena cells, starving them, mating, and isolating the nuclei.

Page 98

Greider draws a hemocytometer, which is a tool to count cells and determine cell concentrations. Greider also writes that she mixes 1 mL of cells with 10 μL formaldehyde for use in the hemocytometer. Formaldehyde is used to kill the cells so they don’t move while using the hemocytometer.

Page 99

May 19, 1984. A timeline for cell matings and nuclei isolation. Greider adds the times 8 am and 10 am to the timeline; Greider probably added these times at a different time than she wrote the rest of the notes on the page.

Page 100

May 20, 1984. Greider details how much to inoculate cells to give a desired cell concentration. Greider counts cells at noon and writes her calculations; however, she later adds that she read the hemocytometer wrong and adds in the correct cell count in blue pen.

Page 101

May 21, 1984. Greider notes cell numbers at different time points. She notes that “cells were too dense” because of the incorrect reading of the hemocytometer, so she decides to dilute the cells.

Page 102

May 20, 1984. Greider details how she spins down the Tetrahymena cells and starves them. Greider checks on them at 5 pm and at 11 pm she counts the cells for mating purposes and spins them down. Greider concludes that she is losing many cells during the spins.

Page 103

May 21, 1984. Greider pools the two preps from May 15, 1984 and May 19, 1984 of pIIb treated with Bal 31 and Hha, does ethanol precipitation and resuspends the pellet in TE buffer. The preps are divided into 3 tubes (one with S1 enzyme added, another with ExoIII enzyme added, and another with λExo added). Greider writes the components of each reaction and the steps after the reaction is done.

Page 104

May 23, 1984. Greider details the composition of a 2x reaction mix. She also attaches a picture of a gel (1% agarose run at 80V) that she ran to make sure the Exo enzymes (ExoIII and λExo) were working. She notes that she didn’t use the S1 enzyme so bands are not as sharp, but she concludes that the enzymes are working “by fuzziness of bands”.

Page 105

May 23, 1984. Greider tests her phenol because she was worried she might be losing DNA because of the phenol she was using in the phenol extraction process. She sets up an experiment to compare it to Beckendorf’s phenol. Greider doesn’t specify further who Beckendorf is, but she did work in Steven K. Beckendorf’s lab at the University of California, Berkeley, so she is probably referring to him. She attaches a picture of a gel (1% agarose run at 80V) at the bottom of the page where she ran her phenol experiment. She concludes that DNA recovery using her phenol is actually good.

Page 106

May 23, 1984. An outline of Greider’s in vitro experiment. It includes using DNA (which presumably is the pIIb treated with Hha and Bal 31 that she had been using previously) in 2x buffer, using the restriction enzyme Xba and running gels. At the bottom of the page she details what componens she used to make stop buffer.

Page 107

May 23, 1984. Continuation of Greider’s outline for her in vitro experiment. She details how she set up 8 reaction mixes (Hot: S1, ExoIII, λExo, control with no DNA; Cold: S1, ExoIII, S1, ExoIII, λExo, control with no DNA). The hot reaction mixes had dCTP and dGTP that had been labeled with the radioactive isotope 32P. DNA is presumably pIIb treated wih Hha and Bal31.

Page 108

May 23, 1984. Nuclei isolation and extract prep protocol. Greider crossed out some numbers and replaced them with others to reflect the actual amounts she used after she wrote the protocol.

Page 109

May 23, 1984. Continuation of the nuclei isolation and extract prep protocol. Sup = supernatant. She mentions using methyl green to check the nuclei. Methyl green stains nuclei green, so nuclei would be readily seen using a microscope. One of her steps consists of saving and storing some of her pellet by freezing it in liquid nitrogen, however Greider notes after that she did not have enough of the pellet to do that. She ends the protocol by setting aside 50 µL of fresh extract for her experiment.

Page 110

May 23, 1984. Two types of extracts are detailed. One with sucrose added and one with no sucrose added to detect which one is more active. Greider notes that the extracts with sucrose added did not pellet nuclei, that the supernatants from + and – sucrose preps did not have nuclei, that the interface from + sucrose prep is viscous which might be due to lysed nuclei, that there are not a lot of whole nuclei seen in the pellet from - sucrose, and that the - sucrose pellet is dirtier than the + sucrose interface.

Page 111

May 23, 1984. Greider notes that she washed the pellet and interface from both – and + sucrose extracts and she re-pelleted them and will “use that final pellet as “Extract” in vitro reaction”. She also describes the + and sucrose pellets. Greider details doing the protocol which includes using the extract and hot and cold dNTPs that she had written about before. She notes that the cold reactions had to undergo phenol extraction again “because they looked goopy”. The last step she notes is cutting with Xba, a restriction enzyme.

Page 112

May 24, 1984. Protocol for how to do the Xba digestion, which is the last step Greider notes in her May 23, 1984 notes. Greider details how she will load two gels (+ or – sucrose).

Page 113

May 25, 1984. Pictures of the two gels (both 2% agarose run at 40V for 17 hours) described on May 24, 1984 are attached. Greider notes that she will cut the gels in half to separate the “hot” and “cold” sides (“hot” side has radioactively labeled nucleotides). She will autoradiagraph the “hot” side and probe the “cold” side with pIIb.

Page 114

May 25, 1984. Greider notes that it seems that the cold reactions (the reactions with unlabeled nucleotides) were cut with Xba, but the hot reactions (the reactions with radioactively labeled nucleotides) were not cut and she decides to maybe phenol extract the hot reactions “after spun column”.

Page 115

May 25, 1984. Autoradiograph of +Sucrose gel exposed for 60 hours. Corresponds to “hot” side of top gel pictured here.

Page 116

May 25, 1984. Autoradiograph of –Sucrose gel exposed for 60 hours. Corresponds to “hot” side of bottom gel pictured here

Page 117

May 25, 1984. + Sucrose gel of cold reactions probed with pIIb. Corresponds to the “cold” side of top gel pictured here

Page 118

Graph done on May 25, 1984.

Page 119

May 25, 1984. Greider describes the components needed to nick translate pIIb. Nick translation involves making “nicks” or deletions in DNA and replacing those with radioactively labeled nucleotides (on this page Greider chooses radioactively labeling dTTP and dATP). That DNA can then be used as a probe, as Greider did with pIIb in the autoradiograph of her cold reactions.

Page 120

May 25, 1984. The effects of the enzymes S1, ExoIII, and λExo on DNA containing telomere sequences. Greider notes that she sees labeling of the (C4A2)40 in the ExoIII and λExo treated DNA. Greider also notes things she wants to do differently in her next experiment.

Page 121

May 25, 1984. Some notes on Greider’s conclusions on the autoradiographs and the blot. One of her conclusions is that the results further confirm that the cold reactions were able to be cut with Xba and the hot reactions were not. This page ends the In Vitro #1 experiment section.

Page 122

Start of In Vitro #2 Experiment notes.

Page 123

On July 2, 1984 Greider cut pIIb with Hha using the components listed. On July 3, 1984 Greider details adding the plasmid pBR 322 in two different buffers (nick translation buffer and TE buffer), doing ethanol precipitation and then adding various components including the restriction enzyme Bam.

Page 124

July 3, 1984. Component volumes needed to make 10x Nick Translation buffer and to make a mix for pBR 322. Greider then details the steps after ethanol precipitation including using spin columns.

Page 125

July 3, 1984. Prep gel of pIIb treated with HhaI is attached. Greider notes she will cut the 3 upper bands and purify them using glass beads.

Page 126

July 5, 1984. Picture of gel (1% agarose) is attached. Greider notes that she is losing a lot of DNA, even with SDS. She also notes that 2-3 ethanol precipitations are effective for nick translation and in vitro experiments. On the left side of the page Greider details cutting the pBR 322 bands and digesting them using the restriction enzyme Bam.

Page 127

July 5, 1984. Greider notes some things she can do differently in future experiments.

Page 128

July 5, 1984. Timeline of Greider’s Tetrahymena cell culture preparation to be used as extracts in her experiment. Greider also details doing duplicate reactions adding pIIb DNA and Bal 31 nuclease, and then splitting the resulting solution into three tubes (S1 enzyme added, ExoIII added, λExo added).

Page 129

July 5, 1984. Greider describes part of the molecular mechanism of exonuclease enzymes and Bal 31.

Page 130

July 6, 1984. Greider details protocol for Bal 31 digests on pIIb. She attaches a picture of a gel (1% agarose) that contains the results of the Bal 31 digests. Greider notes that the “Bal 31 prep looks good”, but she also notes that she lost DNA.

Page 131

July 6, 1984. Preparation of Bal 31 pIIb fragments. Three reaction conditions, each containing a different enzyme (S1, ExoIII, or λExo). A gel (1% agarose) is attached at the bottom of the page showing the results of the ExoIII and λExo reactions at different time points. Greider notes that based on the gel, it looks like ExoIII and λExo reactions are smearing, which is to be expected of single-stranded DNA, therefore it is likely that the enzymes are working.

Page 132

Cell counts at different time points of two mating types of Tetrahymena (4 and 6).

Page 133

Cell counts at different time points of two mating types of Tetrahymena (4 and 6).

Page 134

July 9, 1984. Cell counts at different time points of two mating types of Tetrahymena

Page 135

July 9, 1984: Cell counts at sequential time points to measure the growth of two Tetrahymena mating types (4 and 6). July 10, 1984: Greider counts starving cells.

Page 136

July 10, 1984. Greider details how she grew cells and made extracts following the + sucrose protocol detailed on May 23, 1984. She also writes to do the reactions (S1, ExoIII, λExo) with and without aphidocholin, an inhibitor of DNA polymerases, and with and without radioactively labeled nucleotides (hot and cold). She goes on to detail doing phenol extraction, ethanol precipitation, cutting with Xba, and running 1.2% agarose gels.

Page 137

July 10, 1984. Description of how the gels were loaded.

Page 138

July 9, 1984. Cell counts of two mating types of Tetrahymena IV and VI (4 and 6).

Page 139

July 10, 1984. Notes on what to do for her next experiment.

Page 140

July 11, 1984. Nuclei Isolation and Extract prep protocol using mated vs vegetative Tetrahymena cells. The results of the protocol are detailed at the bottom of the page. The supernatant for both mated and vegetative cells had no nuclei, while the interface for both cell types have nuclei with the mated type interference having more whole nuclei.

Page 141

July 11, 1984. Greider notes storing aliquots of vegetative and mated Tetrayhymena interface in liquid nitrogen.

Page 142

July 10, 1984. Calculations for diluting aphidicholin to desired concentration for reaction. Greider also notes that she will use 1 µL 95% ethanol in 11.5 µL of dd H2O as a control. She added afterwards various changes to her calculations. Greider then describes cell counting for vegetative Tetrahymena cell experiment.

Page 143

July 11, 1984. Counts of mated pairs vs unmated cells. Also Greider added vegetative cell growth notes for Tetrahymena type VI (6).

Page 144

July 10, 1984. Reaction mix components for mated and vegetative cells. The hot reactions have dGTP and dCTP that are radioactively labeled with phosphorus-32, while cold reactions do not have radioactively labeled nucleotides. The reactions also include using ± aphidicholin, a DNA polymerase inhibitor, and DNA treated with the enzymes S1, ExoIII, and λExo. Greider adds later that she forgot to list DTT (dithiothreitol). Aphidicholin is useful because Greider wanted to see if there was a known DNA polymerase that is adding nucleotides to the telomeres or if it is an unknown enzyme that is doing that. The different nucleases she uses are useful to see whether the topology of the DNA affects telomere nucleotide incorporation.

Page 145

July 11, 1984. Components for Xba restriction mix listed to be used by all samples including untreated pIIb fragments.

Page 146

July 12, 1984. Two gels (1.2% agarose) are attached to the bottom of the page. Left gel (①) contains results for mated Tetrahymena cells and right gel (②) contains results from vegetative Tetrahymena cells. The gels correspond to the reactions listed on July 10, 1984. Both gels are divided into hot and cold sides. Hot side includes reactions that included radioactively-labeled nucleotides.

Page 147

Graph done on July 11, 1984.

Page 148

Autoradiogram exposed for 4 days for the hot side of gel pictured on July 12, 1984.

Page 149

July 14, 1984. Notes on autoradiogram of hot side of gels. Greider notes that aphidicholin “did not inhibit non specific labeling”, so perhaps nucelotides are competing with aphidicholin. She also notes that there wasn’t much of a difference between the results for mated and vegetative cells, the differences in labeling of ExoIII, S1, and λExo treated DNA, and that there might be some specificity in the ExoIII treated DNA fragments because its C4A2 fragments (telomere sequence) relative intensities to the internal fragment (1700 bp) is greater in the ExoIII treated DNA with aphidicholin than ExoIII treated DNA with no aphidicholin.

Page 150

July 14, 1984. Continuation of notes on autoradiogram of hot side of gels. Greider notes that the +Aphidicholin samples don’t have fragment labeling in S1 treated DNA samples. She also notes differences in things done in this experiment vs the experiment done on May 25, 1984 and their different results.

Page 151

July 14, 1984. Components and reaction conditions listed for nick translation reaction of pIIb for the July 11, 1984 “cold” blot probe. Nick translation of pIIb results in incorporation of radioactively-labeled nucleotides (32P dTTP and 32P dATP).

Page 152

July 14, 1984. Components listed for prehybridization of cold blots from July 11, 1984 including using denatured salmon sperm DNA. Salmon sperm DNA helps to block non-specific DNA interactions and decrease background noise.

Page 153

Autoradiogram of blot from “cold” side of gel pictured here. Greider writes at the bottom of the autoradiogram “pIIb whole plasmid probe -DNA is dirty! Should have cleaned up!” Page 154

Drawing of what fragments Xba would produce. And notes on what to do next, including ordering rNTPs and adding aphidicholin to extract first.

Page 155

Concentration of dCTP used in experiment.

Page 156

July 16, 1984. Calculations to make acrylamide gel to be used to run the hot side of July 11, 1984 gels. Greider adds later that the gel did not polymerize and that she “should degas gel to aid in polymerization”.

Page 157

July 16, 1984. Notes on running samples from July 11, 1984 using 4% acrylamide gel.

Page 158

July 16, 1984. Components listed to cut pIIb fragments with the restriction enzyme Xba for end labeling.