11/26/18

Rational:

To get an image of the phage found in lysate 8. Also to start DNA extraction by pelleting the phage in lysate 8 and breaking down the free DNA.

Procedure:

• Cleaned lab desk
• Put 10 mL lysate 8 into a tube
• Added 40 ML nuclease mix
• Added 4 mL phage precipitant solution
• Put in shaking incubator for 30 min at 37 C
• Let sit at room temperature for 40 C
• Spun down at 10,000 G for 20 min
• Pulled off teh supernatent
• Froze the remaining liquid and pellet.

Conclusion:

The TEM showed that thr phage had a 106 Mm tail and a 45 Mm head (diameter). Next lab I will do DNA extraction on the phage pellet formed this lab. Nanodrop will also be done on the phage DNA obtained from the DNA extraction.

Fig.4.8 – This image show one of the phage found in lysate 8.

11/19/18

Rational:

To make an EM grid for lysate 8 in order to do a TEM and get an image of the phage in lysate 8.

Procedure:

• Put 15 ML of lysate onto a plate
• Put 2 20 ML drops of water onto the plate and 1 drop of uranyl acetate
• Put the EM grid from A9 darks side down on the lysate and waited 5 min
• Put the grid on the first water drop and waited 2.5 min and repeated for the second water drop
• Put the grid on the uranyl acetate for 1 min
• Put the EM grid in A9 after blotting excess moisture

Conclusion:

Next lab I will do TEM using the EM grid from this lab in order to get an image of the phage that are found in lysate 8. I will also start DNA extraction on lysate 8.

11/16/18

Rational:

To estimate the titer of lysate 8 in order to determine whether a new lysate need to be obtained or not.

Procedure:

• Estimated titer from the spot titer using the 10^-7 dilution
• Titer= (5 pfu/10 ML)(1000 ML/mL)(10^7 ML/mL)

Observations:

• The control plate showed signs of contamination though the spot titer did not
• Most of the spots were cleared
• The spots for 10^-7 and 10^-8 were the only ones that were not cleared
• The 10^-8 spot showed no signs of plaque
• The arthro also seemed to be growing back over the cleared spots
• Titer= 5*10^9 (high titer)

Conclusion:

Since the spot titer showed a high titer then I will do a plaque assay on the 10^-7 dilution in order to calculate the actual titer. I will also do a TEM with this lysate since it is a high titer.

Fig.1.8 – This figure shows the results of the spot titer. The image shows that all of the dilutions up to 10^-7 were cleared. It also shows that there were no plaque for the 10^-8 dilution.

Fig.2.8 – This image shows the contamination found on the control plate for this test.

11/14/18

Rational:

To do a spot test titer from the lysate obtained from last lab’s serial dilution in order to calculate the new lysate’s titer.

Procedure:

• Cleaned lab desk
• Filtered the flooded plates from last lab
• Put 90 ML of PB into 8 microcentrifuge tubes
• Diluted the lysate to get 10^1, 10^-2, … and 10^-8 dilutions
• Put 2 mL of LB broth into a tube
• Added 22.5 ML CaCl2, 2.5 mL TA, and .5 mL arthro
• Poured on plate and waited 10 min
• Put 2 mL of LB broth into a tube for the control
• Added 22.5 ML CaCl2 and 2.5 mL TA
• Poured on plate and waited 10 min
• Spotted the dilutions and lysate onto the plate and waited 10 min
• Put in incubator at 26 C at 4

Conclusion:

The plates from lysate 7 had been completly cleared after 24 hrs so they had been flooded the day before in order to prepare a new lysate. Next lab the titer of this lysate will be determined using the spot titer.

11/12/18

Rational:

To do a plaque assay for three dilutions in order to calculate the titer for lysate 7.

Procedure:

• Cleaned lab desk
• Put 90 ML of PB in to 3 microcentrifuge tubes
• Added 10 ML of the lysate to the first tube (10^-1)
• Added 10 ML of the 10^-1 lysate to the second (10^-2)
• Added 10 ML of the 10^-2 lysate to the second (10^-3)
• Added 10 ML of the lysate and the three dilutions to 4 tubes of .5 mL arthro and waited 10 min
• Put 10 mL of LB broth into a tube
• Added 112.5 ML CaCl2 and added 2 mL the TA mixture to each of the tubes of arthro
• Added 2.5 mL of TA to the dilutions and control
• Poured on plates and waited 15 min
• Put in incubator at 26 C at 3:40

Conclusion:

Next lab the titer of lysate 7 will be determined. If it is a high titer then TEM will be done with the lysate if it is not a new lysate will be prepared in order to get a high titer lysate.

11/9/18

Rational:

To do  a plaque assay in order to determine the titer of the new lysate.

Procedure:

• Cleaned the lab desk
• Filtered  the lysate from the 5 flooded plates
• Put 4 mL of LB broth into a tube
• Added 10 ML of lysate to .5 mL arthro and waited 10 min
• Added 45 ML of CaCl2 to the TA
• Added 2 ml of TA mixture to the .5 mL arthro
• Added 2.5 mL of TA to the control and arthro  and poured on the plates
• Waited 15 min
• Put in the incubator at 26 C at 4

Conclusion:

Next lab we will calculate the titer of the lysate and do a TEM on the lysate if the titer is high enough. If the titer is not high enough then we will get a new lysate with a higher titer.

11/8/18

Rational:

To get a new lysate to run a plaque assay on in order to determine the new titer.

Procedure:

• Added 6 mL of PB to the 5 webbed plates
• Put the plates in the fridge overnight

Observations:

• The plates looked almost cleared, but not quite

Conclusion:

Next lab I will do a plaque assay using the lysate obtained from the five plates that were flooded.

11/7/18

Rational:

To get a lysate that will give a high titer using 5 plates instead of one since the 5 plates prepared last lab are not usable.

Procedure:

• Cleaned lab desk with CiDecon and ethanol
• Put 180 ML of PB to two microcentrifuge tubes
• Added 20 ML of lysate to both
• Added 20 ML of 10^-2 lysate to 5 tubes of .5 mL arthro
• Waited 10 min
• Put 12 mL of LB broth in a tube
• Added 135 ML CaCl2
• Added 2 mL of the TA mixture to the 5 tubes of arthro
• Added 2.5 mL to the 5 tubes and the control and poured them on the plates
• Let the plates sit for 10 min
• Put in the incubator at 26 C at 3:20

Observations:

• Two of the plates slipped though one was still webbed
• One of the plates was completely cleared
• The other two plates were webbed

Conclusion:

Since three of the five plates were unusable the 5 more plates were prepared. Next lab we will flood the 5 plates in order to get a new higher titer lysate. A plaque assay will then be done in order to calculate the new titer.

Fig.2.CW – This image shows one of the plates that slipped, however the majority of the plate still shows the results that were expected.

Fig.3.CW – This image shows one of the plates that was webbed, but did not slip of completely clear the plate.

Fig.4.CW – This image shows the other plate that slipped, though there is no signs of any arthro growing on it.

11/5/18

Rational:

To calculate the titer of the lysate and to take pictures of the phage in the lysate using the TEM microscope.

Procedure:

• Calculated the titer of the lysate
• For the TEM put 20 ML of lysate on a plate
• Put 2 drops of water on the plate and 1 drop of uranyless
• Took an EM grid from A7 and placed it on the lysate drop for 5 min
• Then placed it on the first water drop for 2.5 min and on the second drop for another 2.5 min
• Placed the grid on the uranyless drop for 1 min and then put the sample in the TEM microscope

Observations:

• The titer of the lysate was calculated to be 6.8 * 10^7
• The TEM showed that the tails and heads of the phage were detached

Conclusion:

Since the TEM showed phage that had their tails detached from their heads and a medium titer was used instead of a high titer the TEM will be redone when a high titer lysate is obtained. Next lab we will be flooding the five 10^-2 lysate plates that were prepared by two other group members.

Fig.1.CW – This image shows what could possibly be one of the arthrobacter phage present in the lysate.

10/31/18

Rational:

To record soil G metadata and prepare an enriched lysate for PCR. Also to do a plaque assay with lysate from Claire to determine the titer.

Procedure:

• Cleaned lab desk with CiDecon and ethanol
• Spun down the enriched lysate at 3,000 G for 5 min
• Weighed weigh boat with dry soil

Observations:

• Dry soil with weigh boat- 3.81 g
• Dry soil- 1.82 g
• Percent water- 21.89%
• Sand- 2 mL
  • Percent sand- 20%
• Silt- 4 mL
  • Percent silt- 40%
• Clay- 4 mL
  • Percent clay- 40%
• Soil type- Silty clay

Conclusion:

The soil type for soil G is between clay and clay loam (soil B, C, E, and F), but a little further from soil D (loam). The percent water was also similar to all of the other soil samples. Next lab I will do a plaque assay on the lysate from Claire’s positive sample.