Claire's+Notebook


 * 4/8/14**
 * I have migrated my lab notebook to iPython. You can view my notebook entries and lab notebook [|here]**


 * Link to my evernote:**
 * Claire's notebook**

Extracted 2 samples each of oyster gill and sperm tissue. I extracted different amounts of each sample in order to optimize the protocol. I extracted 30 mg and 10mg from sample 174.gm b (6/2012) as well as 40mg and 20mg of tissue from sample 194.gi (6/27/12). Samples were incubated overnight starting at 4:20pm. All samples were incubated with 1mL of DNAzol and 2.35uL of Proteinase K (100ug/ml). I centrifuged samples at 10,000g for 10 minutes after this incubation and the lysate was collected from all tubes (containing DNA). The leftover pellet containing polysaccharides and insoluble tissue fragments was discarded. 500uL of 100% ethanol was added to the tube, and the tube was inverted and incubated at room temperature for 1 minute. The DNA precipitate was washed twice with 75% ethanol, and spun down briefly to obtain a DNA pellet. DNA was eluted in 200uL of sterile water and pipetted and vortexed vigorously to dissolve the pellet in water. After nanodropping, I noticed that my gill tissue samples likely contain salts or ethanol, so I will do an ethanol precipitation before sequencing.
 * 10/3/12**

media type="custom" key="21030364"

__Total DNA Concentrations:__ 174.gm b A 30mg: 414.11 ng/uL*200uL= 82822ng= 82.822ug 174.gm b B 10mg: 65.34 ng/uL*200uL= 13068ng= 13.068ug 194.gi A 40mg: 362.87 ng/uL*200uL= 72574ng= 72.574ug 194.gi B 20mg: 224.24 ng/uL*200uL= 44848ng= 44.848ug

Test extraction with oyster sperm samples from Mac. Extracted sample 168.gm b and 169.gm a and b which were combined. Samples were incubated for 24 hours starting at 4pm. Both samples were incubated with 500uL of DNAzol and 2.35uL Proteinase K (100ug/ml). The next day, samples were centrifuged at 10,000g for 10 minutes. The lysate collected from centrifugation was extremely viscous, so I added an additional 500uL of DNAzol and incubated for about 10 minutes at room temperature and centrifuged an additional 10 minutes. 500uL of 100% ethanol was added to the tube, the tube was inverted and incubated at room temperature for 3 minutes. The DNA precipitate was removed and washed twice with 1mL of 75% ethanol. At each wash, the DNA was suspended in ethanol, inverted, and stored at room temperature for for a minute to allow DNA to settle. The ethanol was removed by pipetting, and 200uL of sterile water was added to each tube. The resulting DNA was extremely viscous and would not dissolve in water despite continuous vortexing. I was unable to quant the DNA since it was so dense and would not allow pipetting. Next steps: Try same DNAzol extraction with less starting material. Also can try different tissues (gill, sperm) and different methods (Qiagen kit).
 * 9/25/12**
 * DNA extraction**


 * For sequencing require 6 ug's of high mw DNA preferably at ~200ng/uL