NITRATE and PHOSPHATE PROTOCOL

Protocol for Nitrate Testing (High Range 0-30.0 mg/L)
Using the HACH DR 850 Calorimeter
Cadmium reduction method with Nitra Ver 5 Nitrate Powder Pillow

1. Enter the stored program number, PRGM 51, ENTER. The display will show mg/L NO3-N and ZERO icon.
2. Fill a sample cell with 10 mL of sample.
3. Add contents of Nitra Ver 5 Reagent Powder Pillow to the sample cell. Cap sample.
4. PRESS TIMER ENTER. A one-minute reaction period will begin. Shake sample cell VIGOROUSLY until the timer beeps.
5. After the timer beeps, the display will show: 5:00 TIMER 2. PRESS ENTER. A five minute reaction time period will begin.
6. Fill another cell with 10 mL of sample (the blank).
7. Wipe off any fingerprints or liquid
8. Place the blank into the cell holder. Tightly cover the sample cell with the instrument cap.
9. When the timer beeps, press ZERO. The cursor will move to the right, the display should read 0.0 mg/L
10. Place the prepared sample into the cell holder. Cover with the instrument cap.
11. Press Read.

Phosphorus, Reactive (0-2.5 mg/L) SRP (Suspended reactive phosphorous)
Using the HACH DR 850 Calorimeter
Also called Orthophosphate Phos Ver 3 (Absorbic Acid) Method Using Powder Pillows

1. Enter the stored program number for reactive phosphorus, PRESS PRGM. PRESS 79, ENTER. The display will show mg/L, PO4 and the ZERO icon.
2. Fill a sample cell with 10 mL sample.
3. Add the contents of one Phos Ver 3 Phosphate Powder Pillow for 10 mL sample. Shake for 15 seconds.
4. Press TIMER ENTER
5. Fill another sampled cell with 10 mL of sample
6. Place the blank into the cell holder. Tightly cover the sample cell with the instrument cap. PRESS ZERO. The cursor will move and the display will show 0.00 mg/L PO4.
7. After the timer beeps, place the prepared sample into the cell holder. Cover the sample cell. PRESS READ. The cursor will move to the right, the result in mg/L phosphate (PO4 -3) will be displayed.

Protocol: DNA Extraction from Water Sample

Sampling
1. Samples were taken from Island Beach State Park, NJ.
2. On the bayside 4 sterile bags and two water bottles were collected at a depth of 30cm below the surface with a total water depth of approximately 75cm.
3. These samples were then stored in a cooler until transferred to a refrigerator on return to the lab. Refrigeration slows down the metabolic processes.
4. At the site tests were conducted for temperature, pH, dissolved oxygen, nitrate content, and organic phosphorous. See those specific protocols.
5. A second set of samples was collected at a bayside estuary at Island Beach State Park, NJ.
6. At the estuary 4 sterile bags and 1 water bottle of water was collected just below the surface.
7. These were also stored in a cooler until return to the lab – approximately 4 hours – and transfer to the refrigerator.
8. Site tests for temperature, pH and dissolved oxygen were conducted. Nineteen hours after collection, tests for nitrate content and organic phosphorous were conducted.

Filtration

1. A clean, DNA-free 1 L side arm Erlenmeyer with a filter frit hooked to a vacuum of approx. 5 psi was used to filter each of the samples.
2. First, a 1.2 ml Millipore filter was used. As the filtrate slowed this was replaced with another 1.2 ml filter until 5 or 6 filters were collected. (Note: a ml of water typically contain 1 to 6 million bacterial cells). These filters were used to trap particle-bound bacteria and the “bigger” bacteria.
3. The filtrate that was obtained in the above procedure (for both the bay and the estuary samples) was then refiltered using .2ml Millipore filters until 5-6 filters were collected. These filters were used to collect the free-floating bacteria.
4. A quarter section of each filter was removed and frozen to become part of the cryogenic archive at the American Museum of Natural History.

DNA Extraction
Materials:
· DNA extraction buffer =100 mM Tris-HCl [pH 8.0], 100 mM sodium EDTA [pH 8.0],
100 mM sodium phosphate buffer [pH 8.0], 1.5 M NaCl)
· Proteinase K (10mg/ml) – degrades DNAases, RNAases, and proteins
· 20% SDS solution
· Chloroform – hydrophobic; stops foaming
· Isopropanol
· 70% ethanol (EtOH)
· Lysozyme – breaks down glycans
· PVPP - polyvinyl polypyrrolidone; removes humic acid and esters

1. Filters were cut into small pieces and added to a 15 ml centrifuge tube.
2. 50 µl of proteinase K was added to the tube. Additionally, 0.25 g acid washed PVPP and 50 ml lysozyme (20 mg/ml) were added.
3. Place tube in 37°C incubator for 30 minutes. Mix every 5 minutes if a shaking incubator is not available.
4. Add 0.6 ml 20% SDS solution to each tube.
5. Incubate at 65°C for 2 hours. Gently invert tube end to end every 20 minutes.
6. Centrifuge sample at 6000xg for 10 minutes.
7. Transfer supernatant to new sterile, DNA free tube.
8. Resuspend pellet in 2.25 ml DNA extraction buffer and 0.25 ml 20% SDS solution.
9. Incubate for 10 minutes at 65ºC.
10. Repeat steps 7-8-9 one time for a total of 3 centrifugations and 3 (10 min.) incubations. Pool supernatant from repeated steps in a centrifuge tube and mix. Discard the pellet. Seven ml of supernatant will be recovered.
11. An equal volume of chloroform was added to the supernatant and mixed well.
12. Centrifuge at 6000xg for 10 min. (or max. in clinical centrifuge).
13. Remove the top, aqueous layer being careful not to collect emulsion of chloroform and place in DNA-free tube.
14. Add 0.6 ml volume isopropanol to supernatant and let DNA precipitate at room temperature for 1 hour (alternatively, the supernatant-isopropanol could be placed in refrigerator over night).
15. Place 1.5 mls in to ~ 10 microcentrifuge tubes. Centrifuge at max. speed in microcentrifuge for 30 min., room temperature. There should be a faint pellet at the bottom; this is your DNA. Decant off the supernatant and add about 75 ml 70% EtOH to each tube.
16. Vortex vigorously to suspend pellet and transfer DNA to a sterile microcentrifuge tube. Add another 75 ml 70% EtOH to each tube to retrieve residual and add to same tube.
17. Centrifuge in microcentrifuge at 1400xg for 10 min. Remove ethanol. Repeat if necessary.
18. Dry DNA in laminar hood ~ 1 hour or until dry.
19. Resuspend DNA in 50 -100µl sterile distilled (Millipore-DNA free) H2O. Store at -4ºC.

PCR Primer Reactions for Aquatic Isolates

1. Add 100 µL DNA free water to dehydrated DNA extracts (Sigma W4502).
2. Prepare a (+) and (-) control.
(+) with E. coli template
(-) no DNA template

3. If humic acid is suspected in your isolates (observed via brownish color of isolates) you will need to add BSA to the samples.
(BSA acts as a competitive inhibitor for humic acid (HA) preventing HA from attaching to the Taq polymerase and inhibiting the PCR reaction)

Matrix for PCR Trials

4. See note below for order of adding primers, water, isolates and BSA.

A-Add BSA to the bottom of the tube
B- Add R-Reverse primer
Add F-Forward primer
Add T-DNA template (DNA isolates)
C- Water

5. Pulse on centrifuge

6. PCR according to the following cycle specifications:

7. After the PCR reaction is complete, run a gel electrophoresis to determine which of the PCR protocols provided the best extraction/amplification of the DNA.

After the collected bacterial DNA was amplified (PCR I) a gel electrophoresis was conducted to show which of the protocols produced the least contaminated, greatest amount of DNA. It was determined that the following protocols worked best based on the gels:  1A, 2B, 3D, 4E. 1A – 1.2ml estuarian pond   1ml DNA template   0ml BSA (Bovine Serum Albumin)   22ml water 2B - .2ml estuarian pond   5ml DNA template   0ml BSA   18ml water 3D – 1.2ml Barnegat Bay   1ml DNA template   5ml BSA   17ml water 4E - .2ml Barnegat Bay   5ml DNA template   5ml BSA   13ml water 8. Continue with the best protocol for each sample site to ligation and transformation.

Protocol for Transformation of 16 S rDNA Clones from Aquatic Isolates

1. To make the TOPO Cloning Reaction Mix, add together in separate, labeled tubes the following components:

1 µL salt solution (CaCl2)
X nH2O (DNA free)
“x” PCR product (we used 0.5 µL and 2 µL in respective tubes for each site)
5 µL total volume
+1 µL Vector (TOPO Vector, isomerase and plasmid)
6 µL total volume

Mix and incubate at room temperature for 5 minutes.

2. Place mix on ice.
3. Thaw competent cells (DH5).
4. Add 2 µL of TOPO Cloning Reaction Mix to E. coli cell vials.
5. Incubate on ice for 30 minutes.
6. Heat shock for 30 seconds at 42º C (do not shake).
7. Add 250 µL SOC (nutrient broth), cap, invert slowly. Incubate at 37 ºC for 1 hour.
8. Use a sterile glass bacteria spreader to create a lawn of cells on amp + agar plates as follows:
a. Plate 1 use 50 µL reaction
b. Plate 2 use 200 µL reaction

9. Incubate overnight at 37 ºC.

Teacher Notes

· We used PCR Primer Bead containing (Taq polymerase, dNTP’s, Buffers)
· PCR Beads are PuRE Taq Ready to go PCR Beads (27-9559-01 in 0.2 mL tubes, available from Amersham Bioservice, 800 Centennial Ave, Piscataway, NJ, 08855, 732-457-8000)
· Taq polymerase was originally isolated from Thermous aquaticus, a bacteria found in hot springs that acts as a source for polymerase that will not denature at 95ºC, the temperature needed for PCR thermocycle reactions
· PCR product amounts were determined by evaluating the gel bands from DNA extraction.
· For the PCR reaction the amount of water used are determined by creating a total volume of 6 µL
· For the PCR reactions all time and temperature values are critical for the success of the reaction
· During PCR , “finishing of the fragment” means the addition of a poly-A tail to the PCR product which will help the PCR product attach to the plasmid LacZ gene (the LacZ gene is cut to expose T-sticky ends)
· Invitrogen imMedia Amp Blue for lac z-amp recombinant E. coli is available through Invitrogen 760-603-7200).
· Preparation of amp + agar plates(for 40 plates):
Fill 3 flasks with 200 mL distilled water, add the contents of one packet Invitrogen imMedia Amp Blue for lac z-amp recombinant E. coli. to EACH flask.

Picking Colonies/PCR Round II/Electrophoresis to Verify Insertion

1. Pick white colonies on plates; light blue plates are marginal and may be picked if necessary. Light blue plates could be losing their plasmid or it could have been a bad transformation.
2. Circle colonies to be picked – number them and describe color and size of colony. Avoid satellite colonies. These began after the ampicillin was “used” by the other bacteria.
3. How to analyze plate results:
-if there are too few colonies resulting from a procedure then ligate (transform) and replate
-if there are zero colonies – go back to PCR product and vary the amounts then replate and pick these on the next day.
4. Put PCR cocktail into .5ml tubes.
Cocktail (per tube)
1 ml Forward primer (M13-different primer than used in the original PCR reaction)
1 ml Reverse primer (M13)
2.5 ml 10x buffer (containing MgCl2) Mg2+ is the cofactor for Taq
2.5 ml dNTPs
17 ml nH2O
1 ml Taq
25 ml total
5. Pick colonies with the tip of a medium size pipette and place tip in cocktail for a moment, briefly swirl then discard tip. It is not necessary to extract DNA because the E. Coli cells are designed to fall apart easily.
6. Run PCR II amplification (x30 cycles)
Time Temperature(°C) Purpose
5 min 94 Denature
30 s 94 Aneal
15 s 55
30 s 72
5 min 72 Finish
7. Run screening gels of PCR II product to evaluate success of insertion and transformation.
8. Screening gels were run using a 1% TBE buffer with 1g of agarose to 100ml of TBE.

Protocol for Gene Cleaning

1. Transfer the PCR product (into a 1.5 µL tube) that was amplified from E coli colonies screened (+) to contain inserts of 16s rDNA. (+) Plasmids were determined based on gel screening. 7 µL of PCR product were evaluated through electrophoresis identifying inserts of the 16s rDNA.

2. Add 60 µL Sodium iodide. (3 volumes of NaI to product)

3. Add 7 µL glass milk and shake end to end for a little bit. (Glass milk contains a (+) charge that will attach to the DNA aiding in the gene cleaning process; vortex glass milk frequently to keep suspended).

4. Centrifuge on pulse to pellet the glass milk.

5. Remove supernatant (get all liquid!).

6. Add 500 µL of dilute New Wash and resuspend the DNA. (Dilute new wash)

7. Pulse the sample, remove supernatant (pour off then repulse and then remove liquid with micropipetter and decant).

8. Suspend the pellet in sterile H2O.
-if 1st run strong band use 20 µL
-if 1st run faint band use 12 µL

9. Place in a hot water bath at 65 ºC for 10 minutes.

10. Centrifuge for 2 minutes. Can leave supernatant on glass milk until ready for PCR sequencing reaction. Recentrifuge before using.

11. Collect liquid supernatant which will contain DNA.



Perform the sequencing reaction

1. Take a 0.2ml tube. Add 4 ml of liquid containing:
a. dNTPs
b. ddNTPs
c. Big Dye
d. 1 µl water
e. forward primer (either reverse or forward could be used; both are not used because the16s sequence is ~1400bp and the sequencer can only effectively read about 600bp so even if reverse and forward primers were used we would not be able to overlap the fragments)

2. Add 1 µl of gene cleaned product (do not get any of the glass milk).

3. Centrifuge (pulse).

4. Hold at 4°C

Preparation of Sequenced Samples (precipitation)

1. Remove sequencing reaction from the thermal cycler and transfer the contents of the tube to a 0.5 ml tube.
2. Add 65 ?l of 65% isopropanol to the tube – could be done before step 1 (precipitates the reaction).
3. Place in the centrifuge and spin at the max speed for 30 minutes.
4. Remove all the liquid in the tube with a micropipettor and discard the liquid.
5. Add 65 ?l of 75% isopropanol to the tube.
6. Place the tube in the centrifuge and spin for 15 minutes (washes the sequencing reaction).
7. Remove all the liquid in the tube with a micropipettor and discard the liquid.
8. Allow the DNA to dry in the heat block at 45?C.
9. Resuspend the reaction in 1.5 ?l of loading dye/formamide (80?l formamide and 20?l dye).
10. Place tubes in the thermocycler for 3 minutes at 95?C.
11. Immediately place the tube on ice. Allow it to cool for 3 minutes.
12. Load sample in the sequencer.