Friday, June 26, 2009

Woods Hole Oceanographic Institute Marine Microbial Biogeochemistry

I have got some more reading to do:
Main question I'm asking myself: How can I incorporate isotope geochemistry into my project? Secondary question: How do I get myself a job at Woods Hole?

But first- does anyone out there know how to do Unix? ARB is ridiculous!
I am stuck on step #7:

For now, I'll just reminisce about the ocean and re-read the ARB instruction manual.

Tuesday, June 23, 2009

MOBIO experienced User Protocol

Extraction of the water filters that came from the surface waters of the Eastern Twin Mound (Above picture) went well. PCR of the DNA will take place tomorrow. The protocol for yesterday's extraction changed. I used the soil extraction kit instead of the phenol:cholorform protocol from Dr. Vesbach's lab.

DNA Extraction from water sample (using Mo Bio Power Soil Kit catalog #12888)

Sample should have sucrose lysis buffer added in the field and be stored at -80 degrees Celsius. Be sure to do a negative control to ensue sterile technique. Everything added to the sample should also be added to the negative control with exception of the actual sample. All centrifuge spin times are done at 10,000rcf at room temperature.

1. Place sample on ice and allow to thaw completely.
2. Add 60 uL of solution C1 to the Mo Bio Powerbead tubes. Add sample to the tube. Be sure to add as much as possible. Leave room in the top of the tube.
(C1 contains SDS and other disruption agents required for complete cell lysis. In addition to aiding in cell lysis, SDS is an anionic detergent that breaks down fatty acids and lipids associated with the cell membrane of several organisms. It if gets cold, it will form a white precipitate in the bottle. Heating to 60 degrees Celsius will dissolve the SDS and will not harm the SDS or the other disruption agents.)
3. Vortex for 1 minute.
4. Centrifuge for 30 seconds.
5. Pour supternatant to a sterile 2ml microcentrifuge tube.
6. Add 250 ul of solution C2, vortex and incubate at 4 degrees Celsius for 5 minutes.
(Solution C2 contains a reagent to precipitate non-DNA organic and inorganic material including humic substances, cell debris, and proteins; these material can contaminate the DNA purity and inhibit further analysis of the gene in question)
7. Centrifuge for 1 minute.
8. Without touching the pellet transfer 600 ul (or less) of solution to a new sterile 2 ml microtube.
9. Add 200 ul of solution C3, vortex and incubate at 4 degrees Celsius for 5 minutes.
(Solution C3 is a second reagent that removes contaminating organic and inorganic material)
10. Centrifuge for 1 minute.
11. Transfer 750 ul (or less) of solution to a new sterile 2 ml microtube. (Do not touch the pellet)
12. Add 1200 ul of solution C4 and vortex.
(Solution C4 is a saline solution that allows the DNA to attach to the silica inside the Spin Filter.)
13. Transfer 675 ul of the solution to a spin filter and cetrifuge for 1 minture. Decant the flow through (Remove the liquid in the bottom of the spin filter).
14. Repeat step 13 until no solution is left.
15. Add 500 ul of solution C5 and cetrifuge for 30 seconds. Discard the flow through.
(Solution C5 is an ethanol based wash solution. The ethanol cleans the DNA that is attached to the silica filter of any excess salt, humic acid, and other contaminants.)
16. Centrifuge an additional 30 seconds to remove any excess C5 from the silica filter.
17. Transfer the filter to a labeled sterile 2 ml microtube.
18. Add 30 ul of solution C6 to the membrane filter. Be sure to get the solution onto the actual white membrane in teh filter.
(Solution C6 is a sterile elution buffer (10 mM Tris) that wets the membrane and effectively releases the DNA from the membrane.)
19. Centrifuge for 30 seconds.
20. Discard teh filter and store the tube containing DNA at -20 degrees Celsius.

Currently reading:

Stranger in a Strange Land, Robert A. Heinlein

Sunday, June 21, 2009

Back to the Lab

Microbiology Update:
Tomorrow is my first day back in the molecular laboratory. Ian Mcmillan will be showing me how to extract DNA from the filters that I collected cells in two weeks ago. The filters have been sitting in the -80 Celsius freezer since their collection. Below is the protocol written by Justine Hall and Kendra Mitchell. I will be changing it with advisement from Dr. Northup by applying a bead beating method instead of gently 'inverting' the filter after the CTAB and pK are added.
My experiment for this month is to try the bead beating, the gentle inverting technique, and using a MoBio kit on the filters. I hope to determine which method will produce the best results from the waters I am working with.
Geochronology update:
I need to get my travertine samples from this past fall drilled and prepped for analysis the in clean lab. I need the U-series dates for my five samples to complete my cross section of the anticline.

Collecting community biomass from water

Sterivex GP 0.22um Filter unit – Millipore Cat. # SVGP01015.
60 ml syringe with luer lock
3 ml syringe with luer lock
SLB (20mM EDTA, 400mM NaCl, 0.7M sucrose, 50 mM Tris, pH 9.0)

These filters are designed for sterilizing solutions but are also large enough that we have collected biomass from “clear” water and successfully extracted DNA. The more cells that you collect, the better your chance of getting decent DNA. So, maximize the amount of water you filter. For Yellowstone boiling springs, we tried to filter at least a liter of water or until the filter clogged. Be very careful to keep your syringe and filter “sterile” while you are filtering the water. Don’t touch the end of the syringe or any part of the the filter. I used the packaging that both syringe and filter come in to keep everything sterile. You should be able to concentrate on not contaminating your sample because the procedure is very simple.

1. Cleanly open the syringe packaging
2. Suck up 60 ml of spring water. Caution: if your source pool is near or at boiling, the water is likely to boil in the syringe because you’ve lowered the pressure. This will cause very hot water to squirt out of the syringe. Make sure that you point the syringe away from your field mates and away from the pool.
3. Cleanly attach the filter to the syringe. I open the packaging just enough to get the syringe into the packaging and, holding the filter through the packaging, twist the syringe on.
4. Pull the filter and syringe out of the packaging.
5. Push the water through the filter
6. Cleanly remove the filter from the syringe. Again I never touch the filter directly, only through the packaging.
7. Go back to step 2 another 15 times or until the filter clogs
8. Once you have clogged the filter, push all the water out of the filter by sucking air into your syringe and blowing it through the filter.
9. Draw 0.5 ml SLB into a 3ml syringe
10. Very slowly push as much SLB into the filter as you can without it coming out the outflow. We are trying to lyse the cells that are stuck to the filter and preserve their DNA.
11. Leave the 3ml syringe on the inflow to seal that end of the filter
12. Jam a p10 pipette tip on the outflow of the filter. Melt the tip to seal it.
13. Place the whole unit-filter and syringe-in a whirl-pack. Store at –80 until you extract the DNA. We have held the filters at ambient temperature for up to 10 days before freezing.

Extracting from Sterivex filters requires only slight modification of the mocat DNA extraction. I have always treated sterivex as if they were 200 μL samples, which makes it possible to fit all of the reagents in the filter while still getting a good DNA yield. If you have more than 200ul SLB in your filter, you could remove all but 200ul and store the rest at –80 as a backup. All reagent volumes in this protocol are written for a 200 μL sample, but you may have to adjust them if you have a lot of residual water in the filter. This procedure can be performed on aerodisk filters, but it is a lot more tedious and will likely result in low DNA yields due to spillage.

1. CTAB Buffer: 1% CTAB, 0.75 M NaCl, 50 mM Tris pH 8, 10 mM EDTA
2. Proteinase K
3. 20% SDS
4. phenol:chloroform:isoamyl alcohol (25:24:1) or chloroform:isoamyl alcohol (24:1)
5. chloroform
6. 3 M NaOAc
7. absolute ethanol
8. 70% ethanol
9. 10 mM Tris, Filter sterilized

1. Attach a p10 pipet tip to the small end of the filter. Melt the open end of the tip to close it. This is not a perfect fit, so make sure the tip is really on and thoroughly melted closed before adding any reagents.
2. Remove the syringe and add 400 μL CTAB and proteinase K to a final concentration of 100 ug/ml. This must be done SLOWLY as the reagents get bottlenecked at the top of the filter. A bubble may form in the neck of the filter – you can try to pop it or work around it. Replace syringe (can be used to blow reagents into the filter after they are all added).
3. Incubate for 1 h at 60 C. Filters can be rubber-banded to a rotator or you must periodically invert them.
4. Add SDS to a final concentration of 2%. Again, add reagent slowly and watch for bubble formation. Replace syringe.
5. Incubate for 1 h at 60 C.
6. Take syringe off and push all of the air out of it. Replace syringe, turn Sterivex upside down, and suck all of the lysate into the syringe. You may need to remove the pipet tip once all lysate is out of it. Put all lysate into an epi-tube for the rest of the extraction.
7. Extract with an equal volume of phenol:chloroform:IAA (24:24:1) or chloroform:IAA (24:1).
8. Extract twice with an equal volume of chloroform if using phenol:chloroform:IAA.
9. Add 0.1 volumes of 3M NaOAc, invert gently several times, add 2 volumes of 95% ethanol (Note: it may be necessary to split your sample in half to accommodate volumes)
10. Precipitate for 1h to overnight at -20 C
11. Spin for 45 minutes at 14 K rpm
12. Wash with 70% ethanol, spin for 30 minutes
13. Speed-vac to dry pellet, resuspend in 50 ul of filter sterilized dd water or 10 mM Tris, pH 8.0

Wednesday, June 17, 2009

My first Ragnar Relay

I am running the 1st, 13th, and 25th leg of the Ragnar Relay in Utah this Friday and Saturday. The race starts in Logan and ends in Salt Lake City, Utah. There are a total of 11 team members and 186 miles to cover. Above are the logistics for each of my legs. I'm only doing 20 miles, and my legs are flat or downhill. Woo-hoo!

Sunday, June 14, 2009

NSF Comments

I applied for the NSF Graduate Research Fellowship Program last October. My application was reviewed by a panel of academic experts in the science, mathematics, or engineering discipline that identified in my chosen field of study. These are the comments that were made about three essays written by myself, and three letters of recommendation by my advisor, Dr. Diana Northup, and Dr. Anna-Louise Reysenbach. I rank in the 70th percentile for the range of applicants in the year 2008. I will try again this year, and use these comments to strengthen my personal statement, my future research plans, and my previous research experience. The NSF JAM 2009 Conference also helped me wrap my mind around the specific things that the NSF panels are looking for with these types of applications. Better luck this year!
Overall Assessment of Broader Impacts: Very Good
Explanation to the applicant:

By participating in a variety of undergraduate research opportunities and later participating in various out-reach activities you seve as an effective "scientific ambassador" within your community. Your application could be strengthened by elaborating a little more completely on your professional goals for the future
Overall Assessment of Intellectual Merit: Very Good
Explanation to the applicant:

Your previous research record is tied logically to your current investigation. Your research plan to compare microbial communities in marine and terrestrial settings is both worthwhile and original. Your application can be strengthened by providing more information on why/how you would expect these communities to compare.
Overall Assessment of Intellectual Merit: Good
Explanation to the applicant:

You are certainly a motivated, talented student and one who has moved forward in spite of facing adverse challenges. It is noted coming from a rural reservation area and accomplishing what you have thus far - is commendable. It is clear from your previous research experiences you have the passion, motivation and interdisciplinary understanding to plan and conduct a research project. You have built on previous research experiences to develop an original research project. It is noted in your letter of reference your ability to work independently as well as on a team and your development as a student. There is value to your research findings for a number of disciplines. You have worked to build your capacity in a number of science areas - to fully undertake the research proposal. It seems you have access to necessary resources - and have taken advantage of the STEM enrichment opportunities at the University of New Mexico (LSAMP, McNair etc.) to ensure your intellectual capacity in your Ph.D. You have drive and passion and these are critical traits for a researcher - partnered with your cultural background - there exists opportunities to support the oral history/Indigenous knowledge of the Navajo Nation. Your proposal would be strengthened by a stronger academic record as well as assurance you have access to sufficient resources.

Overall Assessment of Broader Impacts: Very Good
Explanation to the applicant:

You have been an active participant in programming focused on increasing the number of under-represented students and professionals in STEM. You have followed the pathway and it is noted you are maintaining active involvement in the next generation of students moving forward. The research experiences in England and the Atlantic Ocean sound to have created a motivation to pursue a Ph.D. It is understood you have developed a more global context to the broader impact of your research. You have developed a strong dissemination plan and have worked to present your research at local and national levels. Your proposal would be strengthened and more competitive with additional letters of support. Your letters of reference should provide specific examples of your research work, aptitude for and skills acquired during the experience.

Friday, June 5, 2009

Second day at Tierra Amarilla

The second day of sampling went well. Two springs, Iron Spring and Blowhole were successfully sampled. Three more springs were supposed to be sampled from, but were dry. On a trip to the anticline in November, these springs were still active. The change in water level is very interesting. I will return in July and again in the fall to see if the water level rises or recedes.
I will bring a YSI monitor out to the field in July (hopefully) to record fluctuations in pH, T, DO, and conductivity.
There are two springs located along the core of the anticline that have low water levels. The water is about 5m down from the surface. We think that the water in these holes is mainly from runoff and rain. The CO2 is still bubbling in them, but the DO, pH, T, and Conductivity are drastically different from the other springs (which we know are sourced from the subsurface) and much more similar to rain water. There might be a gaseous leak that is not correlated with the aquifer?
Photos from top: View from the trail of the southern terminus of the anticline, Blowhole spring, Minas Tirith, Iron spring, Mini Minas Tirith, Pressure Ridge with the Nacimiento in the background. All photos are courtesy of Ara Kooser. He has more photos at his flicker site:
Currently reading:
Redox Processes and Water Quality of Selected Principal Aquifer Systems, McMahon and Chapelle, Groundwater vol. 46, no.2, 2008

Thursday, June 4, 2009

San Ysidro

Sampled seven spring this week. Looked at pH, T, Conductivity, Total Dissolved Solids, Dissolved oxygen, and collected water samples for major anion and cation analyses, along with alkalinity, nutrient content, and inorganic and organic content. I also collected microbial cells in sterivex filters for later extraction at variable depths in two "key" springs.