Laboratory Notebook 2: In Vitro 8-25, 1984 - 1985

Ran certain nucleotide sequences, like G4T2 (one hot and one reacted) through the enzyme Kinase. The hot G4T2 sequence that ran through the gel the reactants didn’t seem to work properly. Tried purifying the nucleotides which then resulted in faded bands. In the end, she decided to use the DNA from yeast. She extracted the oligonucleotides and spun the it to create a pellet. However, the pellet didn’t form. But was more successful the second time. In the notebook, she drew a diagram of pellet DNA that was run through a gel. The DNA had created long ladders of DNA. Found that within her gels the tRNA was present in most of her recent gels. In previous experiments like IV 14 and IV 15, she utilized repeated oligonuciotide yeast experiments and combined them with the Kinase enzyme.

Removed nuclei from cell cultures of Tetrahymena as before. However this time she used a crude cell extraction interface.It also includes up on how to extract and clean nuclei for future use. The gels she ran used different ratios of TTP and ATP. This seemed to created different reactions in the nucleotides being used. The pattern of Thymadine and Guanine changed in the sequence.

She tried a separate experiment using the previous cells extracted from the Tetrahymena and also using it with Yeast and Aphid. Makes a mix of the extracts and ran one of them in a Urea gel and the other into a Formaldehyde gel. In the Urea gel, the Aphids only produced Guanine and Thymidine. The Tetra had produced Adenonine and Thymadine. Images provided in 72 and 73.

IV 20-23- She ran a pellet DNA test, however, was supposed to boil the extract so that it produced duplicate samples of DNA. Decided to continue the experiment and runs two separate reactions with and without Oligonucleotides. This created three separate sets of duplicates. She graphed the curves of the d duplicates.

Set up the reaction again but boiled it as she was supposed to before. Counted the samples afterwards and ran a statistical analysis as she did for the previous experiment. And created a graph as before. With each reactant that was graphed, she then determined the nucleotide sequence with mostly Thymidine and Guanine. The graphs show that by boiling the oligonucleotides, you end up with a lower count and having them in the reactants.

IV 24- Varied the amount of hot/cold Thymadine and hot/cold Guanine and the Oligonucleotides. Set up tubes and diluted each set. Ran two sequence gels and graphed them with the oligonucleotide decreasing in count.

IV 25- loaded the gels and ran it. Found that she accidentally ran the gel backwards,which made results difficult to interpret.