October 31

October 31 2018 Webbing a Plate- Soil C

Rationale: The purpose of this lab is to calculate the amount of lysate needed to create a webbed plate and run a plaque assay.

Description of Procedures:

  1. The workstation was cleaned using aseptic technique and an ethanol burner was lit to create an aseptic zone.
  2. The plaques on the 10^-1 dilution plaque assay were counted and 92 were found.
  3. The diameter of the plate was measured and found to be 86mm, and the radius was 43mm. The diameter of 10 plaques was found and the average diameter was calculated to be 2.4mm.
  4. Next, the titer of the plate was found to be 9.2 x 10^4
  5. The area of the plate was calculated to be 1849 pi, and the area of the plaque was found to be 1.44 pi.
  6. Next, the area of the plate was divided by the area of a plaque to calculate how many plaques would be needed to web the plate. This calculation determined that 1284.03 plaques would be needed.
  7. The amount of lysate needed to web a plate was then calculated by dividing the number of plaques needed by the titer, and changing the units to ul. It was determined that 14.01 ul of lysate would be needed to web the plate.
  8. 14.01 ul of the lysate was added to 0.5 ml of arthrobacter and allowed to sit for 10 minutes.
  9. Top agar solution was made for three plates. 6 ml of LB broth and 67.5 ul of CaCl2 were added to a tube.
  10. 7.5 ml of 2x TA was added to the tube. 4.5 ml of top agar solution was then added to the 0.5 ml of arthrobacter and poured onto a plate labeled LIP 14.01 ul PA 10-31-18. The rest of the top agar was poured onto a plate labeled LIP LJF 10-31-18 Control.
  11. The plates were then allowed to sit for 10 minutes and then inverted and stored in the incubator until the next lab.
  12. The workstation was cleaned using aseptic technique and materials were properly stored or disposed of.

Observations/Results:

  • Observations:
    • Bubbles formed on the plates
  • Results:
    • Titer of lysate: 9.2 x 10^4
    • Average area of plaques: 1.44 pi
    • Area of plate:  1849 pi
    • Lysate needed to web a plate: 14.01 ul

Interpretations/Next Steps:
The procedure was complete. The next step will be to flood the webbed plate to obtain more lysate.

Category: Lucy Potts | LEAVE A COMMENT
October 31

Lab Day 20: Spot test + Plaque Assay

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.

Category: Soo-Un Jeong | LEAVE A COMMENT
October 31

10/29/18 Gel electrophoresis

Rationale: Perform Gel electrophoresis to test the presence of bacteriophage DNA clusters after performing PCR with three primers.

Procedure: 

 Before the experiment was performed, the workspace was cleaned with both Cidecon and 70% Ethanol. 4omL of TBE and 0.8g of Agarose was added into a 250mL hydro flask. The solution was microwaved for 2 minutes and placed to cool. The solution was then poured onto a plate to solidify. Once the solution solidified, TBE was filled up to the wells, and DNA ladder was placed into the first well. Primers 1, 2, and 3 plus DNA dye solutions were added to the next three wells, next to the DNA ladder. Once the solutions were placed into the wells, the VMR  was plugged in, and it ran for 45 minutes and the results were imaged.

Results from Gel electrophoresis

Observations: 

Placing DNA dye into wells was hard since some of the Primer/DNA dye solutions was not placed into the wells, but diffused into the Gel made. Starting the VMR was hard as well since it was not set correctly/turned on/chords not plugged in correctly.

Conclusions:

Gel electrophoresis was nice to do and this test is faster than running multiple plaque assays/spot tests since no contamination factors can ruin a Gel Electrophoresis. If the experiment comes back to be negative:

  • Collect new soil
  • Perform PCR experiment and alternate the variables.

If the experiment comes back to be positive, perform spot test and plaque assay, knowing that phage is present in the soil sample.

 

Category: Michael Lum, Uncategorized | LEAVE A COMMENT
October 31

Calculating Titer 10/31/18

Rationale: In order to guarantee a webbed plate so we can conduct Transmission Electron Microscopy I have received 1mL of Katherine’s lysate and I will calculate the titer and the amount needed to web a plate. I will place more than the calculated required amount so it will definitely be webbed.

Procedure:

  1. Counted plaques on plate and took average diameter of plaque.
  2. After calculating titer realized that there wasn’t enough lysate to create a webbed plate so plate was flooded.
  3. Added 8mL of phage buffer to plate then parafilmed it and stored at 4ºC overnight.

 

Observations: 514 plaques were formed with 25μL of lysate. This means that the titer is 2.056 * 10^4. The diameter of the plate was 86mm and the average diameter of a plaque was 0.82mm. This means that the amount of pfu needed to web a plate is 1.1*10^4. So the amount of lysate needed to web the plate is 535μL of lysate.

 

Conclusions and Next Steps: Now I must make a plate with 1070μL of lysate in order to guarantee a webbed plate.

Category: Sriram Avirneni | LEAVE A COMMENT
October 29

Results from 10/26 PA and Second Passage Continued (10/28/18)

Results:

No contamination or plaques were observed from the plaque assay performed on 10/26 as shown below.

Rationale:

Since it does not appear a phage was picked previously, two plaques will be picked from the “KEA 10/22 100-1 PA” plate.  To continue the purification process, plaque assays will be performed with a plaque, phage buffer mixture solution made from both the newly picked plaque along with the “KEA 10/22 100-1” mixture.

Procedure:

  1. Once an aseptic zone was established, 100 µL of phage buffer was placed into both microcentrifuge tubes “KEA 10/29 100 A” and “KEA 10/29 100 ”
  2. Used a micropipette tip to touch a plaque from the “KEA 10/22 100-1 PA” plate and then swirled the tip in the “KEA 10/29 100 A” microcentrifuge tube.
  3. Used a micropipette tip to touch a different plaque from the “KEA 10/22 100-1 PA” plate and then swirled the tip in the “KEA 10/29 100 B” microcentrifuge tube.
  4. Both “KEA 10/29 100 A” and “KEA 10/29 100 B” microcentrifuge tubes were vortexed.
  5. 25 µL of “KEA 10/22 100-1”, “KEA 10/29 100 A” , and “KEA 10/29 100 B” mixtures were added to correlated test tubes which already had 0.5 mL of Arthrobacter in them.
  6. 8 mL of LB Broth, 90 µL of CaCl2, and 10 mL of 2X TA were combined into a conical vial.
  7. Transferred and mixed 4.5 mL of the Top Agar (TA) mixture from the conical vial into each test tube.
  8. Each test tube was poured onto their correlating plate, and the remaining 4.5 mL of the TA mixture was poured onto a plate labeled “ML KEA 10/29 Control.”
  9. These plates were placed in the incubator at room temperature.

Observations:

  • The following calculations were performed to determine enough LB Broth, 2X TA, and CaCl2needed for 4 plates.

Original Recipe

 X4

2 mL LB Broth

 8 mL LB Broth
2.5 mL 2X TA

10 mL 2X TA
22.5 μL CaCl2

90 μL CaCl2
  • The plaques circled belowed were picked to make “KEA 10/29 100 A” and “KEA 10/29 100 B” plaque, phage buffer mixtures. To increase the chances a phage was picked, the plaques were picked under a light microscope.

  • When pouring the TA mixture, the TA mixture started to solidify causing bubbles and lumps to form on the plates.

Next Steps:

If there is contamination, a plaque assay with be run again with the same mixtures. If there are plaques, a third passage will be performed. If there are no plaques, a different plaque will be picked.

Category: Kathryn Adkins | LEAVE A COMMENT
October 29

Soil Enrichment and Filtration 10/29/2018

Rationale: Centrifuge and filter contaminated samples and enrich soil to get fresh direct and enriched isolations using filters.

results from the heating and killing bacteria method

The results showed high levels of contamination, but we were advised to centrifuge and re-filter the samples and then plate them

Process: 

Soil Metadata

  • mass of weigh boat and dry soil = 8.651 g
  • mass of weigh boat and wet sample = 14.225 g
  • mass of weigh boat = 2.315 g
  • mass of H2O
    • 14.225 g – 8.651 g = 5.574 g
  • mass of wet soil
    • 14.225 g – 2.315 g = 11.910 g
  • percent water
    • 5.574 / 11.910 * 100% = 46.8% H2O

Re-purification of Lysate

  1. transferred ~1 mL of samples from plate wells into micro-centrifuge tubes
    1. wells with arthro were put into one tube and the wells without arthro were put into another
  2. the tubes were centrifuged for 2 minutes at 13,300 G
  3. supernatant was emptied into a clean dish and then filtered with a syringe filter into two fresh micro-centrifuge tubes
  4. filtered lysates were stored in the fridge

 

Soil Enrichment

  1. a 15 mL conical tube was filled with soil to the 3 mL mark
  2. LB broth was added to the 12 mL mark
  3. mixture was shaken and vortex-ed for ~10 minutes
  4. tube was centrifuged for 5 minutes at 3,000 G
  5. the supernatant was filtered using a syringe filter into two 15 mL conical tubes
    1. One tube was labeled direct and stored in the fridge
    2. 0.5 mL of arthro was added to the other tube (enriched) and it was left in the shaking incubator at 27 °C and 150 RPM

Observations:

The enriched sample had very little lysate in it – only 1.5 mL.

Next Steps:

Perform a spot test with the new direct and enriched isolations, as well as the re-filtered lysates (arthro and no arthro) from the previous week.

Category: Rachel Melone | LEAVE A COMMENT
October 29

Lab Day 19: Filtering

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%

Category: Soo-Un Jeong | LEAVE A COMMENT
October 29

10.29.18 PCR for Claire and Lucy

10.29.18 PCR for Claire and Lucy

Rationale: Since no phage had been obtained from any samples previously run, it was found most pertinent to help with other samples that had more promise of developing a high titer lysate. Therefore, a PCR test was run for samples from Claire and Lucy to help verify the PCR procedure was working properly.

Procedure:

  1. Aseptic zone established
  2. Obtained enriched lysate from LIP Soil Sample C
  3. 1mL enriched lysate from LIP Soil Sample C was added to a microcentrifuge tube
  4. Microcentrifuge tube placed in 37°C container for 10 minutes to boil
  5. Obtained 3 tubes with 12.5μL of Taq Polymerase in them. Tops labeled star 1, star 2, star 3.
  6. Added 4.5μL DDI water to each tube
  7. Added 4μL Primer 1, Primer 2, and Primer 3 to corresponding tubes
  8. Added 2μL of boiled enriched lysate from LIP Soil Sample C and Claire’s sample.
  9. Thermocycled all tubes.
  10. Cleaned and tidied bench.

Results/Observations:

  • No new results regarding soil samples were obtained today after the negative result found on Wednesday 10/24.
  • The enriched sample that was used was slightly pink in color, which is different from the normal pale yellow.
  • Volume of sample added to the PCR tubes appeared to be slightly varied rather than a consistent 2µL. Some drops struggled to fully reach the bottom of the tubes.

Conclusions/Next Steps:

  • The PCR tests run today will serve to show if the PCR procedure is working correctly. Since there have been no positive results using the PCR procedure (other than some positive controls), it was found necessary to run a sample that is known to have phage that was found in the class. After a gel electrophoresis is run on Wednesday 10/31, it will be known whether or not the sample is working correctly.
Category: Henry Burns | LEAVE A COMMENT
October 29

PCR for Claire and Lucy 10.29.18

Rationale:

To conduct a PCR test in order to test the validity of the PCR procedure used by utilizing lysates that have been confirmed positive.

Procedures:

  1. Setup an aseptic zone.
  2. Transferred 1mL each of Claire’s ELF-B and Lucy’s ELF-C to micro test tubes, “Claire” and “Lucy” respectively.
  3. Boiled both “Claire” and “Lucy”.
  4. Added 4.5µL DDI water, 2µL “Claire”, 2µL of “Lucy”, 4µL Primer 1, and 12.5µL of Taq Polymerase to a PCR tube. Labeled with a star and number to indicate Primer type.
  5. Repeated step 6 for Primers 2 and 3.
  6. Thermocycled all tubes.

Observations/Data:

I observed that some of the additions to the microtubes did not immediately make contact and mix with the main body of fluid.

Conclusions/Next-Steps:

The next step will be to conduct a gel electrophoresis test in order to confirm or deny the validity of the PCR procedure by seeing if the tests come back positive as they are expected to be.

Category: Nathan Newton | LEAVE A COMMENT
October 29

October 29 2018 Serial Dilutions- Soil C

Rationale: The purpose of this lab is to perform serial dilutions.

Description of Procedures:

  1. The workstation was cleaned using aseptic technique and an ethanol burner was lit to create an aseptic zone.
  2. 90 ul of phage buffer was added to four tubes.
  3. 10 ul of phage buffer lysate from the picked plaque was added to to the 10^-1 tube. 10 ul of the 10^-1 buffer was then added to the 10^-2 tube. This was done out to a dilution of 10^-4.
  4. 10 ul of each tube was added to 0.5 ml of arthrobacter and allowed to sit for 10 minutes.
  5. 10 ml of LB broth and 112.5 ul of CaCl2 were added to a tube. 12.5 ml of 2x TA were added to the tube.
  6. 4.5 ml of the top agar solution was added to the 0.5 ml of arthrobacter and poured onto plates labeled LIP 10-29-18 PA 10^-1, LIP 10-29-18 PA 10^-2, LIP 10-29-18 PA 10^-3, and LIP 10-19-18 PA 10^-4. The rest of the top agar solution was poured onto a plate labeled LIP 10-29-18 control.
  7. The plates were allowed to sit for 10 minutes and then inverted and stored in the incubator until the next lab.
  8. The workstation was cleaned using aseptic technique and materials were properly stored and disposed of.

Observations:

  • Bottom top agar had not set correctly on some plates.
  • Bubbles seen on plates.

Interpretations/Next Steps:
The procedure was complete. The next step will be to make a webbed plate.

Category: Lucy Potts | LEAVE A COMMENT