10/29/18

Rational:

To collect, filter, and collect metadata on a new soil sample in order to perform PCR and gel elctrophoresis on that soil sample.

Procedure:

• Collected soil 2 ft from a Chestnut Oak in the Earle Courtyard
• Measured trunk circumference 137 cm from the ground
• Measured the tree height and canopy width
• Cleaned lab desk with CiDecon and ethanol
• Put 2 mL of soil G into a tube
• Added 10 mL of LB broth and shook for 15 min
• Weighed empty weigh boat and weigh boat with soil G then put under hood
• Put soil in pH vial and added Di water and shook
• tested the pH
• Centrifuged soil G for 10 min at 3,000 G
• Put 10 mL of soil into a tube
• Added 20 mL of DI water and 3 drops of soil dispersion
• Shook the tube for 45 sec and put under hood
• Filtered soil G and added .5 mL arthro
• Put in shaking incubator at 4:15-2:30 at 26 C

Observations:

• Tree height- 666.9 cm
• Trunk circumference- 44.4 cm
• Canopy width- 167.6 cm
• Weigh boat- 1.99 g
• Weigh boat with soil- 4.32 g
• Wet soil- 2.33 g
• pH- 6.5

Conclusion:

The results for soil F were inconclusive so a new gel elctrophoresis will be done, but another soil sample has been prepared for PCR so that a gel elctrophoresis can be done on it at the same time as soil F. Next lab I will do PCR on soil G.

Fig.1.G – The Chestnut Oak found in the Earle courtyard. Note there are two Chestnut Oaks in close proximity, however the other is significantly more healthy.

Fig.2.G – There are several very yellow leaves as shown in this image.

Fig.3.G – The leaf size and shape of this tree which identifies it as a Chestnut Oak.

Fig.4.G – This image show the pH of soil G is 6.5 as seenby the color of the pH paper.

10/24/18

Rational:

To run the samples from last lab through gel electrophoresis in order to determine whether there are arthrobacter phage present in the soil.

Procedure:

• Cleaned the lab desk with CiDecon and ethanol
• Weighted .72 g agarose powder
• Put 35 mL 1x TBE into a flask
• Added .72 g agarose powder
• Heated until the powder was dissolved and then it was cooled
• Added 1.7 ML EtBr and poured into the gel mold
• Then covered the gel with 1x TBE
• Added 5 ML of DNA ladder to a well
• Added dye to PM1 (primer mix 1)
• Added PM1, PM2, and PM3 to wells
• Turned on electrophoresis and waited 45 minutes

Observations:

• The picture of the gel electrophoresis had a white spot (possibly other DNA) covering the middle of the gel

Conclusion:

The spot in the middle of the image of the gel elctrophoresis prevented the results of the gel electrophoresis from being seen. As a result a new gel electrophoresis will be done next lab. A new soil sample will also be collected and filtered in case the results are negative.

Fig.4.F – This image shows the gel electrophoresis when it started. The dye around the wells is what overflowed out of the well when they were beig filled.

Fig.5.F – This image shows the results from the gel elctrophoresis. The white area seen in the middle may be DNA that leaked from a punctured well or from coming in contact with something else that has DNA on it.

10/22/18

Rational:

To cut and multiply the DNA in the soil F lysate so that it can be tested for arthrobacter phage. Also to record the results for the percent water and soil texture.

Procedure:

• Cleaned lab desk with CiDecon and ethanol
• Centrifuged the enriched lysate at 3,000 G for 5 minutes
• Put the enriched lysate into a microcentrifuge tube
• Put 4 ML of primer mix 1, 2, and 3 into 3 different tubes
• Added 6.5 ML of DDI water to the three tubes
• Added 2 ML of enriched lysate to the tubes
• Prepare 3 negative control tubes
• Put 4 ML of primer mix 1, 2, and 3 into 3 different negative control tubes
• Added 8.5 ML DDI water to the three tubes
• Put the tubes into the thermocycler

Observations:

• Sand- 3.5 mL
  • Percent Sand- 35%
• Silt- 3.5 mL
  • Percent Silt- 35%
• Clay- 3.0 mL
  • Percent Clay- 30%
• Soil Type- Clay Loam
• Dry soil with weigh boat- 9.01 g
• Dry soil- 6.44 g
• Percent Water- 18.17%

Conclusion:

The percent water in soil F was similar to all of the other soil sample except for soil C. The soil type which was clay loam was the same as soil Band E. The soil type was also similar to soil C (clay) and soil D (loam). Next lab I will be running a gel electrophoresis with the samples that I ran PCR on.

10/17/18

Rational:

To filter out soil F after it is collected and enrich it for next lab. Soil metadat will also be collected on soil F for future reference.

Procedure:

• Collected soil from a Post Oak outside of the Business Building
• Cleaned the lab desk with CiDecon and ethanol
• Put 2 ml of soil into a tube
• Added 10 mL of LB broth and shook for 15 min
• Put the tube into the centrifuge at 3,000 G for 5 minutes
• Put 10 mL of soil into a tube
• Added 20 mL of DI water and 3 drops of soil dispersion
• Shook for 30 seconds and then placed the tube under the hood
• Put soil in a pH tube and added DI water
• Shook the tube for 10 seconds and then let it sit for 2 minutes before checking the pH
• Weighed an empty weigh boat and then weighed it with a portion of the soil sample in it
• Put the weigh boat under the hood
• Filtered out the centrifuged soil sample
• Added .5 mL arthro to the lysate
• Put the tube into the shaking incubator at 26 C at 3:40

Observations:

• The tree the soil was collected from had quite a few bare branches and it was lighter than other Post Oaks
• Tree height- 584.8 cm
• Trunk circumference- 41.3 cm
• Canopy width- 170.2 cm
• Soil pH- 6.5
• Empty weigh boat- 2.57 g
• Weigh boat with wet soil- 10.44 g
• Wet soil- 7.87 g

Conclusion:

The soil pH of soil F was the same as soil E which is slightly closer to neutral than the other soil samples. Next lab I will be testing this soil sample for arthrobacter phage. I will also record the results for the soil texture and water percentage.

*Note there was a lot of rain leading up to when soil F was collected so the soil contained more water than usual.

Fig.1.F – This image shows the Post Oak the soil F was collected from. The left side and top of the tree show the most significant absence of leaves.

Fig.2.F – This image show the pH of soil F.

10/15/18

Rational:

The Plaque assay was checked for the presence of arthrobacter phage to determine whether a new soil sample will be collected.

Procedure:

• Cleaned lab desk with CiDecon and ethanol
• Checked the control for contamination and checked the plaque assay for plaque and contamination
• Cleaned lab desk with CiDecon and ethanol

Conclusion:

Both the control and plaque assay for soil E showed no signs of contamination. The plaque assay for soil E showed no signs of any plaque. For next lab I will collect a new soil sample, filter, enrich, and collect soil metadata on it.

Fig.8.E – This image shows the plaque assay for Soil E. The plate shows no signs of contamination or plaque.

Fig.9.E – This image shows the control plate for the plaque assay which shows no signs of contamination.

10/12/18

Rational:

To record results for the soil metadata and to perform a plaque assay on soil E to test for the presence of arthrobacter phage in the soil sample.

Procedure:

• Cleaned the lab desk with CiDecon and ethanol
• Filtered the enriched lysate to get FS lysate
• Put 10 ML FS lysate into .5 mL arthro
• Put 2 mL LB broth into soil E TA and control
• Added 22.5 ML CaCl2 1 M to soil E TA and control
• Added .5 arthro and lysate to soil E TA
• Added 2.5 mL TA to the control and poured on the plate (used maltose agar plate)
• Added 2.5 mL TA to the soil E TA and poured on the plate
• Waited 10 min and then put the plates in the incubator at 26 C at 3:15
• Weighed dry soilfrom last lab and measured the amount of sand silt and clay present in the soil sample

Observations:

• Dry soil and weigh boat-
• Dry soil- 4.02 g
• Percent water- 3.8%
• Sand- 2.5 mL
  • Percent sand- 25%
• Silt- 4.5 mL
  • Percent silt- 45%
• Clay- 3 mL
  • Percent clay- 30%
• Soil type- Clay Loam

Conclusion:

The soil texture for soil E was the same as soil B (clay loam). The percent water for soil E is also very similar to soil B and D, but lower than the percent water found in soil C. Next lab I will check for plaque on my plate and if there are plaque then I will start purification. If there is no plaque or if there is contamination I will do the technique that everyone else who has not found a plaque yet will be doing.

Fig.5.E – This image shows the plate for soil E plaque assay. The yellow circles indicate where bubbles in the top agar ar located.

Fig.6.E – Show the control plate for soil E plaque assay. The yellow circles show the location of bubbles in the control plate’s top agar.

Shows the tube containing a sample of soil E which show the amount of sand, silt, and clay in soil E.

10/10/18

Rational:

To get a new soil sample, collect soil metadata and filter it so that a new plaque assay can be done.

Procedure:

• Collected a new soil sample 2 ft from the base of a Post Oak
• Cleaned the lab desk with CiDecon and ethanol
• Put 2 mL of soil into a tube and added 10 mL LB broth
• Shook the tube for 15 min then centrifuged it at 3000 G for 10 min
• Weighed and empty weigh boat and then weighed it with some of the soil sample in it
• Placed the weigh boat under the hood
• Put 10 mL of the soil sample into a falcon tube
• Added 20 mL DI water and then added 3 drops of soil dispersion
• Shook the tube for 30 sec and then place the tube under the hood
• Put some of the soil sample into a pH vial and added DI water
• Shook the vial for 10 sec and then checked the pH
• Filtered the soil sample that was in the centrifuge and added .5 mL arthro
• Put the enriched lysate into the shaking incubator at 26 C at 4:00

Observations:

• The leaves on the Post Oak were slightly brown and seemed to have been eaten by something
• Tree height- 1,066.8 cm
• Tree trunk circumference- 106.7 cm
• Canopy width- 457.2 cm
• Empty weigh boat- 1.98 g
• Weigh boat with soil sample- 6.93 g
• Wet soil weight- 4.95 g
• pH of soil E- 6.5

Conclusion:

The second plaque assay for soil D showed no plaque so a new soil sample was collected. Soil metadata was also collected and the pH of the sample was 6.5 which is slightly closer to neutral (less acidic) than the other samples. The sample was also filtered and enriched for next lab. Next lab I will do a new plaque assay for the new soil sample and record the results for the soil metadata

Fig.1.E – This image shows the tree that the new soil sample was collected from.

Fig.2.E – Shows a healthy looking leaf from the Post Oak that the soil sample was collected from.

Fig.3.E – The leaves in this image have been eaten by something especially at the bottom of the leaves.

Fig.4.E – Shows the pH of this soil sample is 6.5.

10/8/18

Rational:

To do another plaque assay to look for the presence of arthrobacter phage in a plate that is not contaminated.

Procedure:

• Cleaned lab desk with CiDecon and ethanol
• Added 10 ML FS lysate into .5 mL arthro
• Put 2 mL LB broth into the TA mixture and control
• Added 22.5 CaCl2 1M to TA mixture and control
• Poured .5 ML arthro and lysate into TA mixture
• Put 2 mL TA into the control and poured on the plate
• Put 2.5 mL TA into the TA mixture and poured on the plate
• Waited for 10 min and then put plates in the incubator at 26 C at 3:30-2:30 10/10

Conclusion:

The first plaque assay for soil D was possibly contaminated s the control showed signs of contamination. As a result a new plaque assay was done. Next lab I will check the plate for plaque and if there is plaque then I will start the next step (purification). If there is contamination or no plaque then I will find a new soil sample and filter and collect metadata on it.

Fig.10.D – Shows the plaque assay from 10/5. The clearings show where there were bubbles in the TA and are not plaque.

Fig.11.D – Shows the control plate. The white spots covering the plate indicate that it was contaminated.

10/5/18

Rational:

To do a plaque assay to check for the presence of arthrobacter phage. Also to compare the soil D metadata with the last two soil samples.

Procedure:

• Cleaned lab desk with CiDecon and ethanol
• Weighed enriched lysate- 15.7 g
• Put in the centrifuge for 5 min at 3,000 G
• Weighed weigh boat with dry soil- 3.5 g
  • Dry soil- 1.04 g
  • Percent water- 15.4%
• Percent soil types
  • 4 mL sand- 44.4%
  • 3 mL silt- 33.3%
  • 2 mL clay- 22.2%
• Put 2 mL LB broth in control and soil D TA
• Filtered enriched lysate to get FS lysate
• Put 10 ML FS lysate into .5 mL arthro
• Put 22.5 ML CACl2 1M into the control and soil D TA
• Added arthro and FS lysate to soil D TA
• Added 2.5 mL TA to control and soil D TA
• Poured on plates and waited 10 min
• Put plates in incubator at 26 C at 3:30
• Cleaned lab desk with CiDecon and ethanol

Conclusion:

The percent water is similar to soil B, but significantly lower than soil C. The soil type for soil D is loam which is similar to soil B (clay loam), but a little different than soil C (clay). Next lab I will chack my plaque assay for plaque. If there is no plaque then I will collect a new soil sample. If there is contamination then I will redo the plaque assay. If there is plaque than I will start purification.

Fig.7.D – This is the control plate for soil D plaque assay. This yellow circles indicate the location on bubbles on the plate.

Fig.8.D – This image shows the plate for soil D’s plaque assay and the yellow circles show the location of bubbles on the plate.

Fig.9.D – This image shows the falcon tube which indicates the amount (and percentage) of sand, silt, and clay in a 10 mL sample of soil D.

10/3/18

Rational:

To collect soil metadata to help explain results in the future. Also to filter soil sample D so that a plaque assay can be performed next lab.

Procedure:

• Cleaned lab desk with CiDecon and ethanol
• Added 2 mL of soil to a tube
• Added 10 mL of LB broth                                                                                                                                                           *  it was later discovered that TA was added intead of LB broth
• Put 2 mL of soil into a tube
• Added 10 mL of LB broth
• Shook the tube for 15 min
• Put the tube in the centrifuge for 10 min
• Added 10 mL of soil to a falcon tube
• Added 20 mL DI water
• Added 3 drops of soil dispersion
• Weighed empty weigh boat- 2.46 g
• Weighed weigh boat with wet soil- 3.69 g
  • Weight of wet soil- 1.23 g
• Placed weugh boat under hood
• Put soil in a pH vial and added DI water
• Shook for 10 sec
• Let sit for 2 min
• Checked pH of the soil
  • pH- 6
• Shook soil in the falcon tube for 30 sec
• Placed under hood
• Filtered soil D with a top filter
• Placed in a shaking incubator at 26 C at 3:30-2:30 10/5
• Added arthro to the enriched lysate before placing back in the shaking incubator
• Cleaned lab desk with CiDecon and ethanol

Conclusion:

The soil sample had a pH of 6 like the two soil samples before. Next lab I will do a plaque assay on soil D in order to check for arthrobacter phage. I will also record the results for the soil texture/type and percent water for soil D.

Fig.6.D – Shows the pH of soil sample D is about 6.