Laboratory Notebook: Yeast, Linear, Plasmids, 1988 - 1989

The notebook begins with Greider doing a large scale digest of each plasmid. The E. coli strain that Greider was using was enclosed inside the plasmid and included with it a detailed restriction enzyme map. Grieder then clipped a vector from the linear plasmid and ran it through a gel, which resulted in degraded DNA.

Because of the degraded DNA Greider decided to use cells from a colleague.She harvested them and added DMSO, shich inhibits reactions in the DNA during PCR,and pulsed it with heat and incubated it.

Greider analyzed the results from two 1 kilobase vectors, one cut from the plasmid and the other that was uncut. She ran gels for both, with the resulting gel study on the next page. The cut vectors were cut with restriction enzyme HIND, but Greider believed she should have cut it with Bal 2 or a combination of HIND and BAM. Along with plasmid experiment, Grieder diagrammed out the yeast plasmid PBRC 14 Vector 3 on the next page.

Then Greider recut it with the right restriction enzymes. In the first gel, the cut vectors had Bal 2. In the second gel, the cut vectors had a combination of HIND and BAM. Also conducted several ligation tests on a colleagues cells. Greider harvested the cells, heated, and then incubated them. Lillian, a collague of hers, made an interesting note. Lillian told Greider that placing patch plates and putting them in the cold room might help. There were several discussions on inoculated yeast preps and running them through gels. The E. coli transformants, or cells, were placed in the cold room in a bag. However, unfortunately most of the colonies are blue in the gel which means they don’t have inserts because of restriction enzymes. List of things to do for the week for Yeast Linear Plasmid Project: such as pBR blot, and a southern blot. Grieder also had diagrams of different models of telomeres, plasmids were circularized/linearized/transformed with certain fragments of the plasmids. As well as some internal diagram of telomere 5 prime to 3 prime. In the diagram two things occurred. It is either a lagging strand replicating past a leading strand, thereby completing the telomere molecule. The second is a 3 prime overhang that creates a unreplication. Grieder created a diagrammatic model of what she sees noting that there are two strands, an A strand and a B strand. The telomere in the A strand elongates in B strand. The strand then replicates, with a small amount missing at the end. Grieder noted that the missing section was actually the chromosome shortening, and can be repaired with repair synth. Grieder created a diagram of repairing synthesis would look like.