- Scan Sam's Paper Notebooks (long term project) -
kubu4
- Talk to Emma or Mac about growing algae -
emmats
- add Spring Seminars to Roberts Lab calendar http://www.fish.washington.edu/seminars/ -
sr320 Mar 28, 2011
Reoccurring
- Dishes
- Autoclave any waste (orange bags)
- Reconcile Purchases
- Make sure Calendar is accurate
- Update Notebook daily
3/3,4,8/2011
- reoccurring
3/2/2011
- read qiagen protocol handbook and took notes to prepare for extraction
2/7/2011
- Quantified juvenile salmon (chelex extraction)
- Spectrophotometer Steps:
2. select [Nucleic Acid (DNA)]
3. 2ul of H2O for blank -> keep on [RNA 40]
4. change to [DNA 50] after water
5. 2ul of each sample and 3 trials of each
*used purified H2O for the first "blank" and the control sample for the second "blank"
- 260/280 and 260/230 = ~1.8-2 "clean/pure" DNA
2/1/2011
- incubate/vortex/centrifuge juvenile sockeye salmon to finish chelex protocol:
- -> http://people.bu.edu/pbarber/Web%20Protocols/Protocol2.pdf
- tomorrow: quantify DNA w/ spectro... chelex blank?
1/31/2011
- Reoccurring tasks
1/25/2011
- Edited references and put them back up on google
- Reoccuring
- Began Scanning Sam's Notebook
1/24/2011
- Revised references @ http://goo.gl/B5CxW (Instructions on sheet)
- Reoccuring tasks
- Entered ORF column in this spreadsheet
1/22/2011
- took biosafety class @ http://www.ehs. washington.edu/psotrain/ onlineclass.shtm
1/21/2011
- begin revising references @ http://goo.gl/B5CxW
1/6/2011
DNA Extraction Chelex- Juvenile Sockeye Salmon
| Sockeye Juv. Sample # |
ID# |
Tube Weight (g) |
Tube Weight w/ Tissue (g) |
Tissue Weight (mg) |
| 1 |
0_13_7 |
1.02 |
1.04 |
20 |
| 2 |
0_13_8 |
1.03 |
1.04 |
10 |
| 3 |
0_13_9 |
1.02 |
1.04 |
20 |
- Cut/Weight Sockeye tissue
- Tomorrow: Finish Chelex Protocol
- Finished tagging all 700 pictures on flickr
1/4/2011
- Added Winter Seminar to Roberts Lab Calendar. Be sure to include Abstract and Bio in the Description. Info at http://courses.washington.edu/susfish/schedule.html -
- began tagging the 700 photos on flickr
- reoccurring tasks
12/17/2010
- made 2L of 25x modified TAE -
- made 10L of modified 1x TAE
- cataloged bagged hard clam samples in -80c from FL-3 and B-4
- Found these websites related to sperm isolation:
- http://www.tour2india4health.com/sperm-extraction-treatment-india.htm
- www.pnas.org/content/81/3/857.full.pdf (mentions the process of homoginization under MATERIALS AND METHODS)
12/15/2010
CHELEX PROTOCOL:
http://people.bu.edu/pbarber/Web%20Protocols/Protocol2.pdf
DNA Extraction (Qiagen Kit)
1. 25 mg of tissue in 1.5uL tube.
2. 180 uL of ATL buffer added
3. 20 uL of Proteinase K added, vortexed for ~10 seconds
*Note: Enzymes should NOT be vortexed before use and should be placed in cooler while in use.
4. Samples kept at 55 degree hot plate overnight for lysis (~16h)
1. Samples were taken from 55 degree plate and vortexed for 15 seconds.
2. 200 uL AL buffer added, mixed by pipetting and then vortexed
3. Incubated at 70 degrees for 10 minutes
*Note: precipitate/gelatinous precipitate may form but will dissolve during incubation
4. 200 uL 100% (200 proof) ethanol added, then vortexed twice for 15 sec.
5. Samples loaded onto DNeasy Mini spin column + centrifuged for 1 min @ 8000 rpm.
*Note: Make sure to apply all of precipitate to spin column
6. Place spin column onto new 2 mL collection tube
7. Add 500 mL buffer AW1, centrifuge for 1 min @ 8000 rpm
8. Place spin column in new 2 mL collection tube and add 500 uL buffer AW2, centrifuge for 3 min @ 14,000 rpm to dry membrane
9. Spin columns placed in 1.5 mL centrifuge tube, 100 uL of buffer AE was added to membrane of each, centrifuged for 1 min @8000 rpm.
10. Samples stored at -20 freezer.
Prepared for a DNA extraction using the DNAzol protocol on Oyster Samples for next time.
1. LYSIS \ HOMOGENIZATION: 1 ml DNAzol + 25 - 50 mg tissue, 107 cells or 0.1 ml liquid sample.
2. CENTRIFUGATION (Optional): 10,000 g x 10 minutes.
3. DNA PRECIPITATION: lysate + 0.5 ml 100% ethanol.
4. DNA WASH: 1 ml 75% ethanol (2x).
5. DNA SOLUBILIZATION: 8 mM NaOH or water.
1. LYSIS / HOMOGENIZATION
A. TISSUES. Homogenize tissues in a hand held glass-Teflon homogenizer. Use a loosely fitting homogenizer, with a tolerance greater than 0.1-0.15 mm. Homogenize 25-50 mg tissue in 1 ml of DNAzol by applying as few strokes as possible. Typically, 5-10 strokes are required for complete homogenization. Small amounts (5-10 mg) of soft tissues, such as spleen or brain can be dispersed and lysed by repetitive pipeting with a micropipette. Store the homogenate for 5-10 minutes at room temperature.
(Optional)
Sediment the homogenate for 10 minutes at 10,000 g at 4-25 C. Following centrifugation, transfer the resulting viscous supernatant to a fresh tube.
This step removes insoluble tissue fragments, partially hydrolyzed RNA and excess polysaccharides from the lysate/homogenate. It is required only for the isolation of DNA from tissues such as liver, muscles and most plant tissues containing a large amount of cellular and/or extracellular material and is also recommended for the isolation of RNA-free DNA.
3. DNA PRECIPITATION
Precipitate DNA from the lysate/homogenate by the addition of 0.5 ml of 100% ethanol per 1 ml of DNAzol used for the isolation. Mix samples by inverting tubes 5-8 times and store at room temperature for 1-3 minutes. Make sure that DNAzol and ethanol mix well to form a homogenous solution. DNA should quickly become visible as a cloudy precipitate. Remove the DNA precipitate by spooling with a pipette tip. Swirl the DNA onto the tip and attach it to the tube wall near the top of the tube by gently sliding the DNA off the tip. Alternatively, transfer the DNA to a clean tube. Store the tubes upright for about 1 minute and remove from the bottom of the tubes the remaining lysate/homogenate.
Degraded DNA and small quantities of DNA (< 15 µg) do not spool onto a pipette tip. In this case, sediment the precipitated DNA by centrifugation at 5,000 g for 5 minutes at 4 -25 C.
4. DNA WASH
Wash the DNA precipitate twice with 0.8 - 1.0 ml of 75% ethanol. At each wash, suspend the DNA in ethanol by inverting the tubes 3 - 6 times. Store the tubes vertically for 0.5 - 1 minutes to allow the DNA to settle to the bottom of the tubes and remove ethanol by pipetting or decanting.
If necessary, sediment the DNA pellet at 1,000 g for 1 - 2 min at 4 - 25 C. To further remove contaminants when isolating DNA from tissues, the first ethanol wash can be replaced with wash in a solution containing 70% DNAzol and 30% ethanol.
12/13/2010
- Posted a blog about my experience in the lab so far
- Scanned class evaluations for fish 310 and biology 533 into two separate PDFs
- Began research on sperm isolation from testis
12/9/2010
- Finished the Bacto Agar and added the algae, and put them on
- Cleared out ALL of the old algae from the fume hood
12/7/2010
- Almost finished Bacto Agar
12/6/2010
- Continued to make Bacto Agar
11/29/2010
- Reoccurring tasks
- Prepared to make Bacto Agar
11/16,17,18/2010
- Reoccurring Tasks
11/12/20
- Finished manuscript page
- Changed algae media
11/10/20
- Same as below
11/9/2010
- Finished entering Hansen samples
- Recoccuring tasks
- Edited a paged on our wiki
11/8/2010
- Entered Hansen samples into a catalog
11/4/2010
- Meeting
- Reoccurring tasks
11/3/2010
- Reoccurring tasks
- Edited our website
11/2/2010
- Edited our How to do page
- Reoccurring tasks
10/29/2010
- Toured the basement
10/27 & 10/28/2010
- Reoccurring tasks
- Examined/took pictures of a gel with digested and undigested plasmids
10/26/2010
- Edited one of the wiki pages
10/25/2010
- Learned how to autoclave bio-hazardous materials
- Counted allege, fed the oysters and made a solution that the allege grows in
- Learned how to organize paperwork for the budget
10/20/2010
- Came in for the meeting
10/19/2010
- Relocated references/work cited information from a paper
10/18/2010
First Day!
- Took a tour of the labs and the rest of the building
- Loaded clam samples into a catalog