November 29

11/09/16-Day 23-Spot Titer

Results: Negative- I forgot to add in arthro to my plate.

Purpose: Use highly concentrated liquid phage sample from flooding last lab (11/07) t0 spot test and determine concentration of phage particles in lysate.

Materials:

  • 10^0 plate, lysate, CaCl2, Dextrose, 2x top agar(from 10/17/16), PY broth(from 10/17/16), pipette and micropipette, phage buffer, conical vial, arthro, microcentrifuge tubes

img_0399

Procedure:

  1. Label microcentrifuge tubes control, 10^0, 10^-1 to 10^-8
  2. Pipette 100 μL of PB into the control and 100 μL of filtered lysate into 10^0 tube. Pipette 90 μL of PB  into 10^-1 through 10^-8 tubes.
  3. Transfer 10μL of solution from 10^0 tube into 10^-1 tube. Vortex 10^-1 tube.
  4. Transfer 10μL of solution from 10^-1 tube into 10^-2 tube. Vortex 10^-2 tube. Repeat process to 10^-8 tube.
  5. Prepare agar with 18μL calcium chloride, 10μL dextrose, 1.5mL PY broth,  2mL 2XTA, arthro
  6. Label plate with name, date, Spot titer, level of dilution-split plate into 10 sections- (10^0, 10^-1, 10^-2, etc and control-keep in mind the control is for the phage buffer to distinguish contamination from top agar and phage buffer if it does occur)
  7. Do not forget to perform separate top agar control plate!
  8. Spot10μL of each microcentrifuge tube into corresponding area on spot plate. Allow plate to sit undisturbed and place in incubator.

Observations: Now that my arthro is actually in, I’m feeling confident that I will get at least a high medium titer. This time, I made a checklist of everything I had to add to make my plate so that I wouldn’t forget anything.

Calculations:

Calculations for glucose:

(40%)(x mL)=(.1%)(4mL)

x=10μL

Calculations for calcium chloride:

1000mM(x mL)=(4.5mM)(4mL)

x=18μL

Next steps: Use medium titer to calculate amount of solution per plate needed for high titer.

Storage: incubator, labeled Koka 11/09/16, Spot Titer

Pic of  negative resultsimg_0401

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November 29

11/07/16-Day 22-Spot Titer

Results: Positive! I got a nice webbed plate so I am using my 10^0 plate to flood and perform a spot titer with

Purpose: Flood 10^0 plate to make a highly concentrated liquid phage sample. Then spot test to determine concentration of phage particles in lysate from flooding the plate.

Materials:

  • 10^0 plate, phage buffer, syringe and filter, 15ml conical tube, CaCl2, Dextrose, 2x top agar(from 10/17/16), PY broth(from 10/17/16), pipette and micropipette, phage buffer, conical vial, arthro, microcentrifuge tubes

img_0399

Procedure:

  1. Flood 10^0 plate with 8mL of phage buffer and wait approximately 1 hour, swirling phage buffer gently.
  2. Tilt plate slightly, allowing lysate to pool to one side by placing lid underneath it.
  3. Use syringe to suck up lysate and attach to filter. Depress syringe plunger in 15mL conical tube.
  4. Label microcentrifuge tubes control, 10^0, 10^-1 to 10^-8
  5. Pipette 100 μL of PB into the control and 100 μL of filtered lysate into 10^0 tube. Pipette 90 μL of PB  into 10^-1 through 10^-8 tubes.
  6. Transfer 10μL of solution from 10^0 tube into 10^-1 tube. Vortex 10^-1 tube.
  7. Transfer 10μL of solution from 10^-1 tube into 10^-2 tube. Vortex 10^-2 tube. Repeat process to 10^-8 tube.
  8. Prepare agar with 18μL calcium chloride, 10μL dextrose, 1.5mL PY broth,  2mL 2XTA, arthro
  9. Label plate with name, date, Spot titer, level of dilution-split plate into 10 sections- (10^0, 10^-1, 10^-2, etc and control-keep in mind the control is for the phage buffer to distinguish contamination from top agar and phage buffer if it does occur)
  10. Do not forget to perform separate top agar control plate!
  11. Spot10μL of each microcentrifuge tube into corresponding area on spot plate. Allow plate to sit undisturbed and place in incubator.

Observations: This is my first webbed plate, so I’m feeling confident that I will get at least a high medium titer. All plaques on webbed plate were evenly spread out this time so my technique has improved. When transferring solutions in microcentrifuge tubes, I make sure to see if the pipette tip is submerged into the solution just because 10μL is such as small amount.

Calculations: 

Calculations for glucose:

(40%)(x mL)=(.1%)(4mL)

x=10μL

Calculations for calcium chloride:

1000mM(x mL)=(4.5mM)(4mL)

x=18μL

Next steps: Use medium titer to calculate amount of solution per plate needed for high titer.

Storage: incubator, labeled Koka 11/07/16, Spot Titer

Pics of  positive resultsimg_0400

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November 29

11/21/16 Day 33: Nanodrop and TEM (Soil Sample A)

Observations: I will be using the E1 tube containing my DNA (which is being stored in the freezer) to begin the nanodrop procedure. Since I have also prepped my grid for the TEM, I am able to see my phage today!

Goals: To determine the concentration of DNA in my E1 tube, which I will then use in the restriction procedure.

Materials:

  • DNA extract in the E1 tube
  • D.I. water
  • nanodrop machine
  • elution buffer
  • clean wipe

Procedure (Nanodrop):

  1. Initiate the nanodrop machine using a drop of D.I. water
  2. Blank the machine using 2 microliters of elution buffer
  3. Wipe off the top of the sensor
  4. Pipette 2 microliters of the E1 DNA extract onto the sensor
  5. Close the machine and begin the nanodrop
  6. Clean the sensor with D.I. water
  7. Store the E1 tube back in the freezer

Results:

E1: 102.9 ng/microliter

caroline-addison-e12

This is the graph of the nanodrop for my E1 tube, and I got 102.9 ng/microliter.

This is the result of the TEM. There are three phages visible in the picture, all with their tails intact.

TEM: While looking for my phage under the TEM, I got to see three of my phages in one picture, which was apparently a rare find, especially since they all still had intact tails. The average diameter of my phage head was 35 nm, and the average length for the tails was about 85 nm.

Next Steps: Since my number was relatively high compared to most other numbers, I calculated that I will need 5 microliters of my DNA for the restriction digest. I divided 500 by 102.9 nm/microliter, which resulted in the 5 microliters. After Thanksgiving break I will be making restrictions.

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November 29

Day 31 Re-Spot Test and TEM 11-28-16

Spot Test 11-21-16

Results: Top agar control plate was normal and clear meaning no contamination in the media used. The spot test plate had no visible plaques; however, since the plate was incubated for a week, the arthrobacter may have grown over the lysed spots or another mishap in the procedure that lead to these results. Because of these inconclusive results, the spot test procedure should be repeated in order to make sure I have a high titer lysate.

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Serial Dilution Spot Test 11-28-16

Purpose: To calculate my high titer.

Materials: Agar plates, PY broth, 2x top agar, calcium chloride, dextrose, serological pipette, micropipette, filtered lysate, Arthrobacter, microcentrifuge tubes

Procedure:

  1. Make dilutions of the filtered lysate.
    1. Add 90 microliters of phage buffer into 10 microcentrifuge tubes.
    2. Add 10 microliters of the filtered lysate into this tube to make a 10^-1 lysate.
    3. Add 10 microliters of the 10^-1 lysate into another tube of 10^-2 lysate.
    4. Continue diluting this lysate to 10^-10.
  2. Obtain a tube of .5mL Arthrobacter.
  3. Add 1.5mL PY broth, 10 microliters dextrose, and 18 microliters calcium chloride to this tube.
  4. Add 2mL of 2x top agar to the tube.
  5. Plate this on a labeled spot test plate.
  6. Make a top agar control plate.
  7. After about 12 minutes, spot each dilution on the spot plate.
  8. Spot the 10^0 lysate and phage buffer as a control.
  9. Incubate plates for 48 hours.

Results: Results will be analyzed next lab period. The lysate and dilutions were refrigerated.

TEM Results 11-28-16

Head Length: 50 nm

Tail Length: 110 nm

c4086d49-1c44-407c-8e3d-a5660785fb15

Unofficial TEM Picture of my Bacteriophage

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November 29

Sea Phages Lab Day 32 (11/28) : Restriction Digests (SOIL A EAJ)

Name: Haley Everroad

Goal: To use restriction enzymes to cut the DNA at restriction sites

Materials: phage DNA, restriction enzymes with buffers, 65 ºC heat block, microcentrifuge tubes (5), microcentrifuge, water

Calculations:

500 ηg ÷ 57.9 ηg/µL = 9 µL

Table:

UNCUT BSTE SCAI Eco RI HIND III
DNA (µL) 9 9 9 9 9
Enzyme (µL) – 2.5 2.5 1 1
10X buffer (µL) 2.5 (Eco RI buffer) – – 2.5 2.5
Water (µL)

(up to 25 µL)

 13.5 13.5 13.5 12.5 12.5

Procedure:

  1. Incubate DNA tube at 65 °C for 10 minutes
  2. Label 5 microcentrifuge tubes with numbers 1-5
  3. Add 13.5 μL to tubes 1-3 and add 12.5 µL to tubes 4-5
  4. Remove DNA tube from incubator, quickly plate it on ice, then microcentrifuge tube for less than 1 minute
  5. Add appropriate buffers to appropriate tubes according to table above
  6. Add 9 µL of DNA to all tubes
  7. Add appropriate enzymes to appropriate tubes according to table above
  8. Vortex each tube quickly
  9. Microcentrifuge tubes quickly
  10. Incubate the tubes at 37 ºC for 2 hours
  11. Remove tubes from incubator and place them in the refrigerator for 48 hours

Summary:

For this lab, I calculated how much DNA extraction to use in order to digest 0.5 µg of my phage DNA. Then, I put 9 µL of my DNA extraction into 5 different microcentrifuge tubes. I did not put any enzymes into the first tube, tube 1.  Into tubes 2-5, I put 4 different enzymes to cut my DNA at specific restriction sites. During the next lab, I will be using these restrictions to make a gel.

Storage:

All of my tubes were labeled with my initials and either 1, 2, 3, 4, or 5. I stored them all in the refrigerator.

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November 29

11/18/16 Day 32: DNA Extraction/TEM Prep (Soil Sample A)

Observations: After checking my spot test from 11/16/16, I calculated my high titer using the 10^-5 block which had 21 plaques on it. This gave me a high titer of 4.2 x 10^8. I can finally start with the DNA extraction process and prepare for TEM.

This is the result of my spot test from 11/16/16. I used the 10^-5 block to calculate my high titer, which ended up being 4.2 x 10^8.

This is the result of my spot test from 11/16/16. I used the 10^-5 block to calculate my high titer, which ended up being 4.2 x 10^8.

high-titer

Goals: Perform the DNA extraction and begin the TEM preparation procedure.

Materials:

  • lysate
  • nuclease mix
  • isopropanol
  • DNA clean-up resin
  • filter columns
  • sterilized water
  • uranyl acetate
  • deionized water (DI)
  • lysate
  • copper grid

Procedure (DNA Extraction):

  1. Put on gloves and lab coats
  2. Pipette 1 mL of 11/11/16 lysate into a 15 mL conical vial
  3. Add 4 microliters of nuclease mix to the vial and invert to mix
  4. Incubate at 37 °C for 10 minutes
  5. Add 2 mL of resin to the vial
  6. Mix by inverting for 1 minute
  7. Transfer 1.5 mL of the solution from the vial to two microcentrifuge tubes
  8. Microcentrifuge the two microcentrifuge tubes for 3 minutes
  9. Use a P1000 and a P200 pipette to pull off the clear supernatant and discard this excess solution, leaving the cloudy solution in the tubes
  10. Add 1 mL of isopropanol to each tube
  11. Invert the solution and flick the ends of the tubes to mix
  12. Microcentrifuge for 3 minutes
  13. Repeat steps 9-12, but this time only using the microcentrifuge for 1 minute
  14. Remove supernatant and add 1 mL of isopropanol again, but do not microcentrifuge
  15. Combine contents of both microcentrifuge tubes into one column-syringe
  16. Attach the syringe with the solution to a vacuum syringe filter, added quantitatively
  17. Vacuum filter the solution and place the filter into a new microcentrifuge tube
  18. Microcentrifuge the solution for about 5 minutes to remove excess isopropanol
  19. Place the solution into a 90°C hot bath for 30 seconds to remove the last bit of isopropanol
  20. Elute the phage DNA by applying 50 microliters of elution buffer or sterile water to the columns and let sit for 1 minute
  21. Microcentrifuge for 1 minute
  22. Combine the products from the pair of tubes and label it with the phage name, date, and E1
  23. Repeat steps 20-21 with a new tube, but after combining the liquids into one tube, label it with the phage name, date, and E2
  24. Store both elution tubes in the freezer until ready for nanodrop and restriction digest and gel

Labeling: 11/18/16, Addison, E1-E2

Procedure (TEM Prep):

  1. Pipette a 5 uL drop of lysate onto a piece of parafilm under a hood
  2. Pipette two separate 5 uL drops of DI water onto the same piece of parafilm, following the drop of lysate
  3. Have a TA pipette a 5 uL drop of uranyl acetate onto the parafilm following the other 3 drops
  4. Use tweezers to place the grid—shiny side down—on the lysate drop for about 5 minutes
  5. Use the tweezers to place the grid on the first DI water drop for about a minute to rinse the grid
  6. Use tweezers to move the grid to the second DI water drop for another minute
  7. Use the tweezers to place the grid onto the uranyl acetate drop for 30 seconds
  8. Remove the grid from the drop using the tweezers and place onto a clean edge of the parafilm—shiny side up
  9. Have a TA store the grid

Next Steps: After calculating my high titer, which was 4.2 x 10^8 PFU/mL, I jumped right into the DNA extraction and prepared for the TEM. I was thrilled to be done with dilutions and spot tests, and the DNA extraction was so exciting even though it was a very lengthy procedure. I am looking forward to next class where I will be able to see my phage under the TEM and I will also complete the nanodrop procedure, which will allow me to calculate how much enzyme I will add when I do the restriction.

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November 29

Gaw, 11/16/16, Day 30, AMI Adopted Phage Plate Flooding, Spot Test, and Day 29 Re-Do

Date: 11/16/16

Goal: To flood plate from Day 29, spot test, and re-do Day 29

Materials: serological pipette, phage butter, parafilm, Syringe, lysate, 50 mL conical vial, microcentrifuge tubes, p200 micropipette, serological pipette, PY broth, arthrobacter, 2X PY top agar, 1M calcium chloride, 40% dextrose, filter, p10 micropipette

Methods for plate flooding: 

  1. Pipette 9 mL phage buffer with serological pipette onto all 3 titer plaque assay plates from Day 29
  2. Let plates sit for 1 hour

Methods for spot test and TA control with Caroline:

  1. Filter flooded plates from Day 29 with a filter and syringe into 50 mL conical vial
  2. Pipette 6 mL 2X PY top agar with serological pipette into 50 mL conical vial
  3. Pipette 4.5 mL PY broth with serological pipette into vial
  4. Pipette 30 microliters dextrose with p200 micropipette into vial
  5. Pipette 54 microliters calcium chloride with p200 micropipette into vial
  6. Draw 3.5 mL of top agar solution on 0.5 mL arthrobacter test tube with serological pipette
  7. Pour solution in test tube onto spot test plate labeled with 8 sections
  8. Pour remaining solution in 50 mL conical vial onto control top agar plate
  9. Pipette 100 microliters of filtered lysate into microcentrifuge tube 10^0
  10. Pipette 90 microliters of phage buffer with p200 micropipette into 10^-1 to 10^-8
  11. Pipette 10 microliters lystate with p10 micropipette in 10^-1; vortex
  12. Repeat previous step until 10^-8 is completed
  13. Pipette 5 microliters with p10 micropipette onto squares on spot test
  14. Place plates in incubator

Methods for Day 29 re-do (titer plaque assay): 

  1. Obtain three 0.5 mL arthrobacter test tubes and pour all into 50 mL conical vial
  2. Add 800 microliters of lysate from “CAG 11/14/16 Flooded lysate 2” into vial
  3. Let sit for 15 minutes
  4. Pipette 54 microliters of calcium chloride into vial with p200 micropipette
  5. Pipette 30 microliters dextrose into vial with p200 micropipette
  6. Pipette 6 mL 2X TA into vial with serological pipette
  7. Pipette 4.5 mL PY broth into vial with serological pipette
  8. Draw 3.5 mL of solution three times and distribute to 3 bottom agar plates
  9. Put plate in incubator

Observations, results, data: 

  • The plates were check on 11/15/16; however, the plates looked like there was no arthrobacter in it. However, the arthrobacter was put into the solution that created the plates. After looking more carefully, it was determined the plates were lysed. Looking at the plates under the microscope, there were specks.
  • Alex also had to same problem with her plates, and we both put in close to 1 mL of lysate for the plaque assay.
  • Because the plates were lysed, the plates were flooded, and the lysate was used for spot testing.
  • When filtering the plates after flooding for 1 hour, the lysate vial spilled, and instead of having around 25 mL of lysate, there is only 10 mL of lysate left.
  • New plates were made though, but 800 microliters of lysate was added instead.
  • The control TA plate was shared with Caroline again.
The plaque assay plates looked as if no lysate had been added; however, lysate was added. After observing the plates under the microscope, it was determined that the plates were lysed. When the plates were viewed through the microscope, there were specks on the plates, indicating that the plates were lysed.

The plaque assay plates looked as if no lysate had been added; however, lysate was added. After observing the plates under the microscope, it was determined that the plates were lysed. When the plates were viewed through the microscope, there were specks on the plates, indicating that the plates were lysed.

 

The control TA plate showed no signs of contamination, so the plaque assay plates were unlikely to have contamination.

This is a picture of one of the plaque assay plates. It appears that there are no plaques; however, after looking at the plates through the microscope, it can be determined that the plates are lysed.

Conclusions, interpretations, next steps:

  • The plates were thought to be lysed because so much lysate was added, but it was doubled because on Day 29, the arthrobacter was running low and had issues with latching onto the phage.
  • The spilled lysate may cause problems in the future; however, more plates can be made.
  • The next step is to perform DNA extraction or TEM.

Labels and placements:

  • The microcentrifuge tubes for the spot test are labeled as “CAG 10^-1 to 10^-8 HT2” and placed in the well in the refrigerator.
  • The flooded lysate was labeled “CAG 11/16/16 Flooded lysate 3 and placed in the refrigerator.
  • The flooded plates were placed in the biohazard bin.
  • The control TA plate was labeled as “CAG CMA control TA 11/16/16” and placed in the incubator along with the plaque assay plates labeled as “CAG 11/16/16 AMI Adopted Phage Spot Test” and “CAG 11/16/16 AMI Adopted Phage Plaque Assay 1-3”.
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November 28

Gaw, 11/14/16, Day 29, AMI Adopted Phage Titer Calculation and Plaque Assay

Date: 11/14/16

Goal: To calculate titer from spot test plate from Day 28 and complete plaque assay using calculations

Calculations:

Count: 47 plaques from 10-2

Equation: # of plaques counted * (1/volume of lysate added) * 1000 microliters/1 mL * 10+x = # plaque forming units/mL

47 * (1/5 microliters) * (1000 microliters/1 mL) *  102 =940000 pfu/mL=9.40 * 105 pfu/mL

Diameter of plaque 0.5 mm=0.05 cm
Area of plaque
π(r2)=π(0.025)2=0.001963 cm2
Diameter of plate 8.4 cm
Area of plate π(r2)=π(4.2)2=55.41769 cm2

Area of plate/area of plaque: 55.41769 cm2/0.001963 cm2=28231.12 cm2

28231.12 cm2 * (1/x microliters) * (1000 microliters/1 mL) *  102 =4.00 * 104 pfu/mL

x=3003.3106/102 =30.033 microliters

Materials: lysate, 50 mL conical vial, p1000 micropipette, arthrobacter, phage buffer, 1M calcium chloride, 40% dextrose, 2X top agar, PY broth, bottom agar plates, serological pipette

Methods: 

  1. Obtain three 0.5 mL arthrobacter test tubes and pour into 50 mL conical vial
  2. Add 950 microliters of lysate into each arthrobacter test tube
  3. Let sit for 15 min
  4. Pipette 54 microliters of calcium chloride into vial with p200 micropipette
  5. Pipette 30 microliters dextrose into vial with p200 micropipette
  6. Pipette 6 mL 2X TA into vial with serological pipette
  7. Pipette 4.5 mL PY broth into vial with serological pipette
  8. Draw all of the solution and distribute 3.5 mL into each plate
  9. Place plates in incubator.

Observations, results, data: 

  • The control top agar was not contaminated.
  • The spot test plate did contain many plates on 10^-2, and they were countable.
  • The titer lysate was calculated using the 10^-2 spot.
  • The top agar solution was completed with Caroline as well as the top agar control plate for this day.
  • Even though the calculation for the titer was higher than before, it was not high enough and should be at 10^8 at least.
This picture shows the results of the spot test. While there were many plaques, the titer calculation was not high enough to proceed with DNA extraction or TEM.

This picture shows the results of the spot test. While there were many plaques, the titer calculation was not high enough to proceed with DNA extraction or TEM.

 

This picture shows the control TA plate as well as the spot test plate. There was no sign of contamination on either of the plates.

This picture shows the control TA plate as well as the spot test plate. There was no sign of contamination on either of the plates.

 

Conclusions, interpretations, next steps:

  • In order to ensure the plate is webbed, the lysate amount was multiplied by 10.
  • Also there was not enough arthrobacter, so only three test tubes of arthrobacter were used and 3 plates were used for the plaque assay.
  • The arthrobacter was a little older and had not experienced growth from Saturday.
  • The next step is to flood the plates with phage buffer and perform a spot test to calculate the titer.

Labels and placements:

  • The control TA was placed in the incubator and labeled as “CAG CMA 11/14/16 control TA”.
  • The plaque assay plates were placed in the incubator and labeled as “CAG 11/14/16 AMI Adopted Phage Plaque Assay 1-3”.

 

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November 27

SEA Phage Lab Day 26-28

Day 26: 11/16/16 Calculating Titer, TEM, and Making Webbed Plates

Results: I got plaques on all of my sections from 10^0-10^-8, which means that I have a high titer.

 img_5379 img_5380

 

Calculating Titer:

img_5470

 

TEM:

Purpose: To see my phage.

Materials: high titer lysate, parafilm, sterile water, tweezers, copper grids, 2% uranyl acetate

Procedure:

  1. I transferred 100 microliters of my lysate into a micro-centrifuge tube.
  2. I stretched out a piece of parafilm.
  3. Then I put 10 microliters of the lysate on the left corner of the parafilm.
  4. Then next to it I put 10 microliters of sterile water.
  5. Next to the water, I put another 10 microliters of sterile water.
  6. Lastly, I put 10 microliters of uranyl acetate on the right corner of the parafilm.
  7. After I set up my work area, I put a small circular copper sheet in the lysate for 10 minutes so that the phages would attach to the copper sheet.
  8. After 10 minutes, I transferred the copper sheet to the first drop of water for 2 minutes.
  9. After 2 minutes, I transferred the copper sheet to the second drop of water for 1 minute.
  10. After 1 minute, I transferred the copper sheet to the uranyl acetate for 30 seconds.
  11. Then I dabbed the copper sheet on a piece of paper to get rid of the excess liquid.
  12. Then Jennifer put my copper sheet into the section of the small rectangular box labelled 3B.
  13. Afterwards, I went to the TEM machine to look for my phage.
  14. The man who was looking for the phage said that he could not find anything on my copper sheet.

Next Step: I need to extract DNA from my lysate. If possible, TEM again at the very end of the semester.

 

Making Webbed Plates:

Purpose: To get a lot of highly concentrated lysate.

Materials: lysate, arthro, PyCA broth, Top Agar, dextrose, calcium chloride, seriological pipettes, empty plates

Procedure:

  1. I got five arthro tubes and added 20 micro-liters of my lysate into them.
  2. I let the arthro tubes with the lysate sit for 15 minutes.
  3. Then I got a 50 mL conical tube and added 50 micro-liters of dextrose, 90 micro-liters of calcium chloride, 10 mL of Top Agar, and 7.5 mL of PyCA broth.
  4. Then, I transferred 3.5 mL of the solution into each of the arthro tubes.
  5. Lastly, I poured my arthro tubes into 5 individual empty plates.
  6. I let the plates sit for 10 minutes.
  7. After 10 minutes, I inverted the plates and stored them in the incubator.

 

Day 27: 11/18/16 Flood Plates

Results: This time all of my plates were webbed so I can flood all of them.

img_5393 img_5394 img_5395 img_5396 img_5397 img_5398

Purpose: To make webbed plates that have a high concentrations of plaques from a lysate that we know the titer of.

Materials: lysate, top agar, PyCA broth, calcium chloride, dextrose, arthro, empty plates, seriological pipettes, 50 mL conical vial

Procedure:

  1. I added 8 mL of Phage Buffer into each of my T1-T5 plates.
  2. I then para-filmed each of the plates.
  3. Then I stored the plates in the freezer for two days

Next Step: I have to collect the lysate and the extract DNA.

 

Day 28: 11/21/16 DNA extraction and Nanodrop

Results: I collected and filtered about 32 mL of lysate.

Purpose: To isolate the phage DNA from the phage

Materials: lysate, micro-centrifuge tubes, centrifuge machine, nuclease, DNA cleanup resin, 80% ethanol

Procedure: 

  1. I transferred 1 mL of lysate into a micro-centrifuge tube.
  2. I added 4 micro-liters of nuclease into the 1 mL of lysate in the micro-centrifuge tube.
  3. I then put the tube in the incubator for 10 minutes.
  4. After 10 minutes, I added 2 mL of DNA cleanup resin (guanidinium thiocyanate) into the centrifuge tube with the 1 mL of lysate.
  5. Then I transferred about 1.5 mL of the solution into two different micro-centrifuge tubes.
  6. Then, I centrifuged the tubes for 3 minutes.
  7. Then I used a pipette to pull up the supernatant and leave only the pellet in the individual tubes.
  8. Then I added 1 mL of 80% isopropanol to the pellets in each of the individual centrifuge tubes.
  9. Lastly I inverted the tubes and lightly flicked the tips so that the pellets would make their way to the bottom of the tubes.
  10. Then I centrifuged the tubes for another 3 minutes.
  11. Repeat steps 6-9.
  12. Add one mL of the isopropanol into the individual tubes, but do not centrifuge it this time.
  13. Invert the tubes and lightly flick them so that the pellets go to the bottom of the tubes.
  14. Then attach the tubes to a vacuum syringe filters.
  15. Then put the tubes in a water bath at 90 degrees Celsius for 30 seconds so that any excess isopropanol will evaporate.
  16. After 30 seconds, centrifuge the tubes again.
  17. Then to elute the phage, Lathan added 50 micro-liters of extremely sterile water into each of the tubes.
  18. Then micro-centrifuge the tubes for one minute.
  19. Then transfer the liquid from one tube to the other so that all the solution is in one tube. Then I labeled it with E1, the date, and No Show (my phage’s name).
  20. Next, I repeated steps 17-19 with a new tube.
  21. Then, I transferred the liquid from one tube to the other so that all the solution is in one tube. This time I labeled the tube with E2, the date, and No Show (my phage’s name).
  22. Then I took my tubes to get them nanodropped with Jennifer.
  23. My nanodrop result was 105.0 nanograms/liter.
  24. After nanodropping, I stored my E1 and E2 tubes in the freezer.

Next Step: Enzyme Restriction

 

 

 

 

 

Category: Navya's Notebook | LEAVE A COMMENT
November 27

Gaw, 11/09/16, Day 28, AMI Adopted Phage Plate Flooding and Spot Test

Date: 11/9/16

Goal: To flood plates from Day 27 and perform a spot test with 100-108  dilutions

Materials: phage buffer, filter, syringe, 50 mL conical vial, p1000 micropipette, arthrobacter, phage buffer, 1M calcium chloride, 40% dextrose, 2X top agar, PY broth, bottom agar plates, serological pipette, microcentrifuge tubes

Methods for flooding: 

  1. Pipette 8 mL phage buffer with serological pipette onto all five medium titer plaque assay plates from Day 27
  2. Let plates sit for 1.5 hours

Methods for spot test:

  1. Filter flooded plates with a filter and syringe into 50 mL conical vial
  2. Pipette 5 mL 2X PY top agar with serological pipette into 50 mL conical vial
  3. Pipette 3.75 mL PY broth with serological pipette into vial
  4. Pipette 25 microliters dextrose with p200 micropipette into vial
  5. Pipette 45 microliters calcium chloride with p200 micropipette into vial
  6. Draw 3.5 mL of top agar solution on 0.5 mL arthrobacter test tube
  7. Pour solution in test tube onto spot test plate
  8. Pour remaining solution in 50 mL conical vial onto control top agar plate
  9. Pipette 90 microliters of phage buffer into all 8 microcentrifuge tubes with p200 micropipette
  10. Pipette 100 microliters of filtered lysate into microcentrifuge tube 10^0 with p200 micropipette
  11. Pipette 10 microliters of solution into 10^0 to 10^-1; vortex
  12. Repeat previous step until through 10^-8 is completed
  13. Pipette 5 microliters onto squares on spot test
  14. Place plates in incubator

Observations, results, data:

  • The control TA plate was completed with Caroline again.
  • The plates were not lysed, but there were enough plaques to flood.
  • The plaques on each of the plates looked similar.
  • The plates were flooded for 1.5 hours which may affect lysate and calculations because the plates before were flooded for 48 hours.
The five plaque assay plates had enough plaques on them for flooding. They did not have any contamination on them.

The five plaque assay plates had enough plaques on them for flooding. They did not have any contamination on them.

 

The control TA plate from 11/7/16 had no contamination.

The control TA plate from 11/7/16 had no contamination.

Conclusions, interpretations, next steps:

  • The next step is to calculate from the number of plaques on the spot test to see if a high titer has been achieved. If a high titer is achieved, then the next step would be to go through the TEM process and DNA extraction. Otherwise, the calculation should be used to complete another plaque assay.

Labels and placements:

  • The spot test plate was labeled as “CAG 11/9/16 AMI Adopted Phage Spot Test” and placed in the incubator.
  • The control TA plate was labeled “EAJ CAG 11/9/16 control TA” and placed in the incubator.
  • The microcentrifuge tubes were labeled “CAG HT 10^0-10^-8” and placed in the wells in the refrigerator.
Category: Christina's Notebook | LEAVE A COMMENT