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.


DNA EXTRACTION FROM STERIVEX FILTERS
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.

Solutions
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

Procedure:
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