Rationale

Webbed the plate with the donor’s 10^0 purified lysate so that nxt lab day, the plate can be flooded. Donor’s calculations of adding 524 microliters of lysate did not full web the plate, so 550 microliters was added instead.

Detailed Procedure

1. Took 550 microliters of filtered enriched lysate and added to 400 microliters of Athro. Sat for 15 mins
2. Took 4.2 mL LB Broth, 45 microliters of 1M CaCl2, and 5 mL of 2X TA into a 50 mL vial.
3. Took half of solution from step 2 and poured into control plate
4. Took other half and added solution from step 1 and poured into plaque assay plate
5. Both plates sat for 15 mins and was inverted into the incubator.

Conclusion/Results/Next steps

Contamination from previous lab day on the control plate, but had plaque present. Plaque from personal plate seem to be bigger in size compared to donor’s plaque. During open lab, results from today’s webbing will be determined soon. The webbed plate was sliding, so the plate was not inverted in the incubator. If plate seems to be webbed enough, the plate can be flooded on open lab.

contaminated control plate

KEA’s results from previous lab.

Rationale

Redo plaque assay from lab day 20 due to contamination of control plate. Instead of using enriched filtered lysate from donor, used purified filtered enriched lysate of 10^0 for the plaque assay.

Detailed Procedure

1. Took 45 microliters of filtered enriched lysate and added to 400 microliters of Athro. Sat for 15 mins
2. Took 4.2 mL LB Broth, 45 microliters of 1M CaCl2, and 5 mL of 2X TA into a 50 mL vial.
3. Took half of solution from step 2 and poured into control plate
4. Took other half and added solution from step 1 and poured into plaque assay plate
5. Both plates sat for 15 mins and was inverted into the incubator.

Conclusion/Results/Next steps

Spot test results from Soil F was negative, so there will be no need to run a PCR or gel electrophoresis on Soil F. To prevent contamination in the plaque assay, new LB Broth and 2X TA was used and labeled group number on them. In the next lab day, if there is no contamination on the control plate, then I will start calculations to web the plate, since the lysate used was already purified.

Rationale

Performed a spot test from the newly enriched soil lysate to test whether or not group 6 as a phage. Performed a plaque assay by using another classmate’s enriched lysate sample which does have phage. I had to perform the plaque assay because I need to be able to start creating a high titer soon.

Detailed Procedure: Spot Test

1. Took two vials labeled as 1X and 2X.
2. For the 1X vial, took 2.5 mL of 2X TA, 2 mL LB Broth, and 23 microliters of 1M CaCl2.
3. Poured 1X vial onto plate labeled as control.
4. For the 2X vial, took 5.0 mL 2X TA, 4.0 mL of LB Broth, 4.5 microliters of 1M CaCl2, and 1.0 mL of Arthro.
5. Took half of the 2X vial and poured equally onto two separate experiment plates.
6. Waited 15 min for plates to solidify
7. Took 10 microliters of each sample and PB and spot tested onto designated plates.

Detailed Procedure: Plaque Assay

1. Took 10 microliters of filtered enriched lysate and added to 0.5 mL Arthro. Sat for 15 mins.
2. During 15 mins, took 4.0 mL LB Broth, 45 microliters of 1M CaCl2, and 5.0 mL of 2X TA into separate 50 mL vial. Mixed through pipetting.
3. Used sterile pipette and took half of solution from step 3 into another separate 15 mL vial. Labeled as control.
4. After 15 mins were done, took solution from step 2 and mixed through pipetting into previous 50 mL vial.
5. Took both vials into water bath and waited for plate to be ready.
6. Labeled plates as “Plaque Assay” and Plaque Assay Control.”
7. Took control vial and poured into control plate and same with other plate+vial.
8. Waited 15 mins to solidify.

Conclusion

On the control plate for the plaque assay, the mixture did not cover the entire plate so it solidified unequally on the plate. Until next lab day, group 6 will check results on the spot test from the new enriched soil and start picking the plaque and creating a high titer from the plaque assay.

Rationale

Obtain results from rest of soil metadata procedures and filter enriched lysate. New procedure of heating the lysate instead of syringe filter. One lysate turned green, so syringe filtering on this lab day might help for future testing.

Detailed Procedure

1. Took dropper and inserted 1 mL of enriched lysate into each of the two microcentrifuge vials.
2. Weighed both vials and centrifuged for 2 mins.
3. Poured the vials of the supernatant onto a petri dish lid, used a syringe filter, and inserted into another two separate microcentrifuge vials.
4. Labeled both vials and stored in the fridge.

Conclusions/Next Steps

One of the lysates turned green while the others did not, despite performing the same procedures. To make sure the lysate was filtered properly, group 6 used a syringe filter to ensure all bacteria is filtered out. Lab day 19 took up a lot of time and did not have much time to perform a spot test/plaque assay. Next lab day, group 6 will all perform a spot test together with one control plate.

Soil Metadata Results

• % water= 46.8%

Rationale

Begin soil metadata to determine percent water, pH, and type of soil collected. Soil washing and enrichment will be done in order to continue from testing through PCR.

Detailed Procedure: Soil Metadata

1. Weighed plate and added soil. Recorded both plate and plate +soil
2. Filled soil up to 10 mL mark in a 50 mL vial and filled rest with D.I water up to 30 mL mark.
3. Added 3 drops of soil dispenser solution and shook for 30 secs
4. Left to rest on rack for 48 hours.
5. Pinched soil into pH vial and filled rest with D.I water and shook for 10 secs
6. Left to rest for 2 mins
7. Pinched off 1 inch of pH paper and dipped into pH vial for 45 secs.
8. Recorded pH of soil

Detailed Procedure: Enrichment

1. Filled soil in 15 mL vial up to 2 mL mark
2. Filled LB Broth into vial from Lab Day 14 up to 12 mL mark in aseptic zone
3. Shook using vortex for 10 mins.
4. Weighed vials and centrifuged for 5 mins
5. Took dropper and poured supernatant into 3 separate wells around halfway.
6. Heated up supernatant at 55 degrees for 5 mins and 60 degrees for 5 mins with shaker-thermostat
7. Took 100 microliters of Arthro into 2 of the 3 wells.
8. Incubated it and shook in the machine.

Results metadata

• pH= 6.5

Conclusion/Next Steps

Group members decided to heat up the supernatant to kill of any bacteria and let it cool down before adding the Arthro. This was a experimental way of making enriched lysate without manually filtering the supernatant. In the next lab day, group members will run PCR.

Rationale

Make the gel for gel electrophoresis. Run a gel electrophoresis to see any sign of plaque from DNA RSM and DNA SJ.

Detailed Procedure

• Took 40 mL of TBE into a flask along with 0.8 g Agarose.
• Microwaved the flask for a bit to heat up.
• Chilled down to warm temperature.
• Added Ethidium Bromide into flask.
• Poured flask contents onto gel tray.
• Let it sat for a few mins to solidify and turned cloudy.
• Put gel tray into gel box and poured TBE just enough to cover the solidified gel.
• Took 10 microliters of Sample 1,2,3 into separate wells.
• Took 5 microliters of ladder into separate wells.
• Took 10 microliters of control (sample 4) into separate gel box
• Ran gel electrophoresis for around 40 mins.
• Took gel off with gloves and place on top of aluminum foil and placed gel onto tray.
• Inserted tray into BioRad Gel Doc EZ Imager to show image of results onto computer.

Conclusion/Next Steps

The image of the gel electrophoresis showed that there is no sign of plaque in both soil sample from SJ and RSM. The next steps is to adopt a plaque from a different group and hopefully continue with picking a plaque.

Rationale

Obtain rest of soil metadata to determine soil type, and percentage water. Start PCR process for enriched lysate from Lab Day 15 to determine if there is a present of plaque and whether or not if it can be tested through spot test/plaque assay in the future.

Detailed Procedure

1. Took 5 mL of unfiltered enriched lysate into a 15 mL vial and centrifuged it for 5 mins
2. Took 1 mL after centrifuge into a centrifuge tube. Labeled as S.J. Heated it up
3. Took 4 tubes that contained Taq polymerase+dNTP.
4. In first tube, PM1 (primer mixer) and 1 microliter of both DNA were added
5. In second tube, PM2 and 1 microliter of both DNA were added
6. In third tube, PM3 and 1 microliter of both DNA were added
7. In fourth tube, PM1, 1 microliter of positive control DNA, and 1 microliter of water were added.
8. All four tubes were labeled with group symbol and numbers and ran cycles

Soil Metadata Results

• plate after 48 hrs= 7.03
• percentage of water= 83.67%
• Sand= 42.42%
• Silt= 30.30%
• Clay= 27.2%

Conclusion

Soil E was determined to be a sand type soil. After the cycle for the 4 tubes are done and put into the fridge for next lab day, group 6 will be able to make gels to see and compare the DNA and determine which one of us have plaque (if present.) If it seems that there might be a hint that there is plaque, then spot test and plaque assay will be performed in the future.

Rationale

Complete enrichment process of new collected soil and results from soil metadata from new soil.

Detailed Procedure: Soil Metadata

1. Weighed plate and added soil. Recorded both plate and plate +soil
2. Filled soil up to 10 mL mark in a 50 mL vial and filled rest with D.I water up to 30 mL mark.
3. Added 3 drops of soil dispenser solution and shook for 30 secs
4. Left to rest on rack for 48 hours.
5. Pinched soil into pH vial and filled rest with D.I water and shook for 10 secs
6. Left to rest for 2 mins
7. Pinched off 1 inch of pH paper and dipped into pH vial for 45 secs.
8. Recorded pH of soil

Detailed Procedure: Enrichment

1. Filled soil in 15 mL vial up to 2 mL mark
2. Filled LB Broth into vial from Lab Day 14 up to 12 mL mark in aseptic zone
3. Shook using vortex for 10 mins.
4. Weighed vials and centrifuged for 10 mins
5. Syringe filtered supernatant (7.5 mL into 50 mL vial and 2 mL into 15 mL vial)
6. Poured 0.5 mL Arthro into 50 mL vial

Results

• weight of plate= 2.06 g
• weight of plate +soil= 8.00g
• ph of soil= 6
• weight of vial before centrifuge= 20.03g

Conclusions

When 48 hours pass, then the type of soil collected will be determined and will know the percentage of water, sand, silt, and clay. After incubation of enriched lysate, group 6 will be able to do spot test and plaque assay of the each new soil and check to see if there are any presence of plaque.

Rationale

Check to see if any plaque remains. If not, why/how does our results mean to the group question. Find new soil. Control plate from Lab day 13 was not contaminated, but showed negative results from plaque assay plate. Other group members did spot test and had negative results and did not have contaminated control plate

Detailed Procedure

• Group 6 dug up new soil from a red oak in front of the LL Sams.
• Soil was dug up around 2 feet away from the tree, near the roots.
• No more plastic bags were present, soil was added to a glove

Conclusion

New soil’s metadata must be done by next lab day to determine which type of soil we collected, and what pH. Soil enrichment will also be done by next lab day. If there is no plaque present, then group 6 will have to adopt a plaque from another group.

Rationale

Perform plaque assay from Soil D without any trace of contamination.  Two members from group 6 did spot test while one did plaque assay. Each test had an individual control plate.

Detailed Procedure

1. Syringe filtered enriched lysate.
2. Took 10 microliters of filtered enriched lysate and added to 0.5 mL Arthro. Sat for 15 mins.
3. During 15 mins, took 4.0 mL LB Broth, 45 microliters of 1M CaCl2, and 5.0 mL of 2X TA into separate 50 mL vial. Mixed through pipetting.
4. Used sterile pipette and took half of solution from step 3 into another separate 15 mL vial. Labeled as control.
5. After 15 mins were done, took solution from step 2 and mixed through pipetting into previous 50 mL vial.
6. Took both vials into water bath and waited for plate to be ready.
7. Labeled plates as “Plaque Assay” and Plaque Assay Control.”
8. Took control vial and poured into control plate and same with other plate+vial.
9. Waited 15 mins to solidify.

Conclusion/observations

In the beginning, the unfiltered enriched lysate had arthro all over the tube which was because the vial was not taken out during the past 4 days. Due to more experience, prevention of contamination technique were better (accidental spills, not being in aseptic zone.) Control plate had no bubbles and did not have problems sliding. Experiment plate had two bubbles and continued to slide even after 15 mins were done. Both plates could not be inverted in the incubator. In the next lab day, future steps would be to check for any signs of plaque. Since within the group, both spot test and plaque assay was performed, so if no plagues were found, then a new soil will be tested next. if there is time this week.