Pryes,+Karissa

BIO 360 Zebrafish Genetics Lab Notebook Karissa Pryes

Lab 1: Overview of Transgenesis Strategy, Genomic DNA Isolation, and Bioinformatics Lab Partner: Katie Haupt

Thursday, September 1, 2011

1. Female zebrafish was anesthetized in 200 mg/L tricaine and decapitated. Caudal fin, head, and scales were removed via scalpel, and fish was cut into smaller pieces. 2. Using a pre-cooled mortar and pestle (stored in -80* freezer), fish pieces were frozen with liquid nitrogen and pulverized into a powder. When pieces started to thaw, they were refrozen with liquid nitrogen until a nice crusty powder. 3. The tissue powder was added to the cell lysis solution, and placed on a rocking platform for approximately 45 minutes. 4. Some larger fish tissues had settled to the bottom of the tube, and we used a pipette to remove the solution suspended above the chunks. This was then carefully layered under 18mL og 190 proof molecular biology grade ethanol. 5. Katie and I created a Shepherd's Crook with a glass pasteur pipette and carefully removed the DNA from the tube. We allowed it to dry slightly, washed in fresh EtOH, and stored in TE in 4*.

Lab 2: Genomic DNA Characterization and PCR Lab Partner: Katie Haupt

Tuesday, September 6, 2011

1. Prepared a 0.5% agarose gel, adding GelStar. We loaded 5 uL into the lanes of the gel. Lane1: High Range Ladder Lane 2: Our isolated DNA sample Lane 3: Known DNA sample (control)

It appears that our lane is very dim, and that our ladder rungs are a little blurred. Maybe the DNA segments are breaking down? And as for our isolated sample, maybe not fully rehydrated before run through, or an issue with storage?

__**Primer Designs: 9.8.11 for the SHHA gene (chr 7)**__ LEFT PRIMER: TTGAACAAACCTCGTGTGGA RIGHT PRIMER: CCACAAGCAAGCGACATAAA

(Dr. Balza, will you please let me know if you can't open this? Thanks!)