Discovery of Ryadel
Isolation Protocol 5.1 – Collecting Environmental Samples
-GPS taken via iPhone.
*The original GPS corrdinents were corrected due to inaccuracy.
| Sample | 1. Lake Kirby Abilene TX | 2. Under rotten hay bail Stephenville TX | 3. Home Depot Granbury TX | 4. Base of tree near LDS Stake Center Weatherford TX |
| Collector Name | Travis Miller | Travis Miller | Travis Miller | Travis Miller |
| Date of Collection |
Sep 4, 2017 |
Sep 4, 2017 |
Sep 4, 2017 |
Sep 4, 2017 |
| Sample Type (Soil/Water) | top layer rocky -1/2 down clay-creamy grey color | sand, clay, silt | Peaty | clay, silt |
| Location Description (compost, lake bed, etc) | low amounts of vegetation. High amount of carp feeding | Overgrown old garden, sold underneath rotting hay bails falling apart. | Kellogg Potting Mix Premium Mix for Outdoor Containers | Base of small tree a lot of thick grass covering soil |
| General Location (forest, subdivision, etc) | Shore side of rocky lake Kirby, | Back yard of a remote subdivision | Stacked on a pallet of soil in the garden section of Home Depot, Out side in the direct sun light | near Weatherford High School |
| Specific Location (GPS coordinates) | 32°22’11.7″N 99°43’35.0″W | 32°16’43.6″N 98°08’52.1″W | 32°25’57.0″N 97°46’55.2″W | 32°43’20.3″N 97°48’32.8″W |
| Sample Depth | 1in water 1 in soil | 2 in | 1 in within bag | 2 in |
| Ambient Temperature | 88F | 92F | 93F | 92F
|
Sep 6
Isolation Protocol 5.2 Direct isolation
Protocol was followed, no changes or modifications occurred.
Observations
Direct – Sample #2
8mL of liquid observed
2 layers of soil
top layer dark
bottom layer light
after 10 mixtures x 2000 centrifuge – 3 layers
new layer is light on top
Sep 6
Isolation Protocol 5.3 Plaque Assay
Day 1
protocol followed, only modification was increased time in incubator
sample sat undisturbed for 7 minutes
allowed for a 20 minute for agar solidification
placed in incubator 3pm 37C
Day 2
Direct Isolation pulled after 96 hours incubation and placed in fridge
Sep 6
Isolation Protocol 5.5 Enriched Isolation
Both samples #3 and #4 were tested under Enriched conditions
#3 observations – 35 mL Enriched solution
floating particles
liquid is cloudy and dark brown
two observable soil layers
top layer – near black
bottom layer – dark brown with light brown
After 10 minutes centrifuge x2000 – gray color particle at bottom formed
#3 filtration – 25 mL
observable yellow tint
#4 observations 37.5 mL Enriched Solution
3 layers formed, grass floating in mixture
top layer – gray material with mix of roots and grass
middle layer – layer of light and dark material
bottom layer – combination of light and dark
#4 filtration – 25 mL
faint tint of yellow
Isolation Protocol 5.6 Spot Test
First spot test completed, procedure was followed. Second spot test preformed procedure was modified
Direct Sample placed #1 spot of 9 spot agar plate
Enriched #3 placed on both the 5 and 7 spot
48 hours incubating – non turbid, yellow tint, all collection of light yellow debris/bacteria
Enriched #4 48 hours incubating, turbid, creamy yellow, small yellow debris present
Two plates were spotted.
First plate
1-3 spots were #3
4-6 spots were control
7-9 spots were #4
After 48 hours, 24 in the incubator and 24 in the fridge, both plates shared evidence of phage. Plate 1 which contained only enriched and control showed phage in sections 1-2-4-5-7-8 and 9-6.
Plate #2 was needed due to a spot error, I forgot to add Direct Isolation sample on plate #1 for spot test. Placed in incubator at the same time as Plate #1, it was also taken out and placed in the fridge as well at the same time.. However, prior to incubation and upon cleaning my workstation I bumped the plate and fell into the floor. The lid did come off which may cause a contamination or a shift in the spots made. Dr. Edwards stated not to worry and to carry on with the experiment. Plate #2 was labeled similar to #1 with 9 sections, section 1 and 3 were spotted with Direct Isolation. A control was placed on section 5 and sample #3 at section 7 and sample #4 was placed in section 9. Sections 2,4,6,8 were all left empty to prevent cross contamination.
After 48 hours, 24 in incubator and 24 in fridge phage was present in direct sections 1 and 3. #3 sample showed evidence of phage in sections 4-5-7-8. This may have been due to the drop. Sample #4 showed no sign of phage growth or progress. Splatter dots were also visible throughout the plate, also attributed to the drop.
Potential mess-ups of class and corrections to be made.
a lot of shifted spots
-to correct problem we all change protocol to 30 minute wait time as opposed to 20 minutes, this will allow more time to set and dry as well as soak in
– Dr. Edwards is testing incubator to ensure incubator is plumb
– to help prevent shift we will also only spot 5mL not 10 as directed
Spot Test 2
Plate labeled in 9 sections.
Section 1 – Direct
Section 3 – sample #3
Section 5 – control
Section 7 & 9 – sample #4
Sample #4 was placed in two sections due to previous spot test. I was curious to know if #4 could actually yield a phage since Direct and #3 sample produced phage from the last spot test, I gave #4 sample two spots.
#4 sample did not yield any phage, Direct and #3 samples once again did yield sign of phage.
Sep 20
Purification Protocol 6.1 Plaque Assay for Purification
Purification Protocol 6.2 Serial Dilution (Direct isolation)
Isolation Protocol 5.4 Picking Plaque
Purification Protocol 6.3 Collecting Plate Lysates
– allowed several colleagues to pick #3 smaple, sample shared with Dannielle, Miranda and Musgrave
-Sample #3 was given to Miranda to carry on into the SEA-PHAGE program
plague picked placed in 1.5mL tube with 100mL of buffer
placed 90 uL of buffer in six 1.5 centrifuge tubes
direct isolation sample picked 10 uL placed in 10^-1 vortex 5 seconds
10^-1 extracted 10 uL placed into the 10^-2 vortex continued steps until 10^-6
gathered six 250 uL of bacteria inoculated each tube of bacteria with the centrifuge tube diluted phage
top agar -3 mL into tube with bacteria and phage
plate onto agar allow to sit 20-30 mins start time 1030-1055 am
Sep 22
Purification Protocol 6.2 Serial Dilutions
Observations over Sept 20th procedure pulled plaque assays 24 hours after incubation placed in fridge
small sign of phage present in 10^-1, 10^-2, 10^-3 no sign of phage in 10^-4, 10^-5, 10^-6
Dr. Pierce informed my dilutions looked good but perhaps my phage needed more incubation time because it was so small it was called “slow grower” placed back in incubator 25th of Sept 10 am left until 9 am Sept 27th
results on serial dilutions
10^-1 hard to see visible virus bacteria
10^-2 virus apparent in bacterial carpet extremely webbed will use 10^-2 for webbed plate collectedly lysate protocol 6.3
10^-3 virus still present appears to look like swiss cheese
10^-4 prior to another 2 days of incubation there is no visible virus now 10^-4 is countable
10^-5 no virus present prior to last incubation cycle 10^-5 showed signs of isolated virus to a single plaque
10^-6 no visible sign of phage no more incubation will be tested because web plate and isolated virus have been achieved
Sept 27
Purification Protocol 6.3 Collecting Plate Lysates
all protocol was followed as directed
web plate was used on 10^-2 dilution finished flooding at 945 am
at 3 pm 5.5 mL recovered from flooded plate
plaque assay 3 mL of top agar 250 mL bacteria mixed to create bacterial carpet let sit 20 mins during that time all serial dilution were made all the way from 10^-1 to 10^-8
spots were made with each dilution and a control with a total of 9 dilutions being tested
spots 10^-1, 10^-2, 10^-3, 10^-4 showed visible spots after an incubation time of 26 hours
speaking to Dr. Edwards he instructed me the plate incubated too long and re-do spot checking every 6 hours
Oct 2
Purification Protocol 6.4 Spot Titer
spot titer used previous serial dilutions spotted plate with 10^-1 to 10^-8 as well as the control
allowed plate to sit for 30 mins once spotted to allow for absorption and then placed in incubator at 1040 am will check again within 6 hours also at 430 pm
returned at 430 pm no visible sign of any bacteria or phage will allow to sit over night and check early morning
Oct 3
Purification Protocol 6.5 Full Plate Titer
dilutions 10^-4, 10^-5, 10^-6
10^-5 showed trace amounts of phage present
10^-4, 10^-6 full plate titers will be made along with 10^-5 to better determine what dilution to full plate titer is most sufficient
plates were still slightly cold from fridge in order to have a better plate skin contact was used to warm up the plastic
plaque assay made at 10 am placed in incubator at 1020 am
Oct 4
after another 24 hours at exactly 910 am plates pulled for observation no growth or phage detected in 10^-4, 10^-5, 10^-6
plates returned to incubator at 950 am another round of incubation
Oct 5
all plates pulled at 920 am after another 24 hours of incubation totaling incubation so far for full plate titer 1 day 22 hours 55 mins
plates 10^-4, 10^-5, 10^-6 are now within counting
10^-4 counted 23
10^-5 counted 10
10^-6 counted 2
instructed by Dr. Edward to allow another cycle of incubation
placed in incubator Oct 5th at 1030 am
extracted plates Oct 6th 1 pm
Oct 6
8 mL phage buffer placed on agar for flooding at 140 pm allowed to sit until 420 pm tome which letting sat 2 hours 40 mins
was only able to extract 3.5 mL of 8 mL
loss of buffer could be due to dry agar absorbing liquid for rehydration another possibly for loss would be filter absorption
after another round of incubation 10^-4 dilution yielded 40 plaques this number will be used to calculate a PFU per mL
estimated plaques on the full plate to create webbed plate would be roughly 4000 plaques per agar
40 pfu / (.01 mL) (1e-4) – – 40 pfu / (1e-6 mL) = 40e-6 = 4e-7 pfu/mL
Oct 9
Protocol 7.1 – Making webbed plate from a lysate of known Titer
Estimated 4000 plaques per plate
volume needed mL lysate = 4000/ 4e-7 pfu/mL
1e-4 mL lysate
1e-4 * 1000 = .1 uL
Webbed plates
.1 uL calculated to be ideal amount for proper webbed plate
It is ideal to test 10x more and 10x less than the amount calculated of lysate to determine if calculating are accurate.
In order to produce enough lysate for analysis it is recommended to have 8-10 mL.
Using a total of 6 plates, 2 plates per dilution.
Dilutions being .01 uL, .1uL, and 1uL
Full plate titer dilution of 10^-4
- 10 uL used to create a .1uL and .01uL for webbed plate
- plaque assay, several dilutions and webbed plate
- 6 plates used 2 per concentration
- 9uL added to tubed, 1uL added to 10^-4 to produce a 1uL dilution
- transferred in normal plaque assay step added to 3mL of top agar and plated
- plating completed at 135pm allowed to sit for 20 minutes for solidification before placing in incubator
- started incubation at 2 pm
Oct 10
plates extracted form incubator at 340 pm for a total incubation time of 25 hours 40 minutes. All plates contain phage.
Oct 11
1uL concentration has the most potential to create a webbed plate
plate left out at room temperature from 340pm to 920 am, this allowed for growth but at a slower rate as opposed to the incubator. Total incubation time for plates
1 day 1 hr 40 min in incubator + 17 hrs 40 min = total growth time 1 day 18 hours 20 minutes
Oct 12
Flooded webbed plated of the 1uL sample used collecting lysate protocol to collect total lysate from flooded plated. 14mL collected lysate, placed in fridge
Oct 17
Dr. Edwards and Dr. Pierce pleased with progress, suggested to do another spot test and full plate titer to verify results.
Followed protocol of spot titer and serial dilution protocol
Oct 18
Dilutions 10^-5 and 10^-6 both best based on countability, pulled at 1015 am
Oct 20
Full plates of 10^-5 and 10^-6 used for plate, however, no plates were available at this time.
Oct 23
Full plate titer on 10^-4, 10^-5, 10^-6 plated
retested the serial dilution
plaque assay
10 uL into 250 uL of bacteria incubation started at 1050 am
Oct 24
plates taken out of incubator at 1210 pm for incubation time of 25 hours 20 minutes
phage present on all dilutions, 10^-4 dilution appears to be the best webbed.
Originally 10^-2 dilution was the best webbed plates it appears to have pushed back the dilution to 10^-4
Oct 25
Procedure 9.1 Phage DNA Extraction
procedure followed as directed in Phage Discovery Guid
Nov 6
Qubit ds DNA Analysis kit
2 .5mL tubes
1 master mix 1.5mL tube
Labeled tube 1 and 2 with master
Dilute 1:200 of Quibit buffer
– add 796 uL buffer into tube master mix
– added 4 uL reagent to same tube
-10uL standard in new tube
- added 10 uL standard into tube 2
- add 190 uL master mix to each, mix well
-add 2uL DNA sample in new tube
-add 198uL mix to tube
vortex 4 tubes -> 2 standards
put samples in Quibit reader
Quibit reader calculated DNA to be 99ng/uL
Nov 6
Restriction Enzyme
| .5 g | mL | 1000 uL |
| 99 ug | 1mL |
= 5.05 uL
Began incubation at 1229 pm, 10 minutes at 65˚ C
25uL H2O added to all 6 tubes along with 2.5uL reaction buffer
| 1 | 2 | 3 | 4 | 5 | 6 |
| BamHi | ClaI | EcoRI | Hae III | Hindi III | Sal I |
| red | green | clear | green | blue | red |
| 3.1 | cut smart | EcoRI Buffer | cut smart | 2.1 | 3.1 |
incubate at 37˚ C for 60 minutes
126pm -> 226 pm
Agar Gel
.8% gel
1% = 1g/100mL
.8g/100mL -> .4g/50mL = 400mg/50mL
8% = x needed / 100mL -> .8*50mL/100 = .4g
Buffer 1x dilute TBE
Final 300mL
30mL buffer 5mL buffer
270mL H2O 45 mL H2O
EtBa
(300mL)(.5ug/mL EtBa) = x 10mg/mL
actual ager = .4003g
2uL EtBa into gel (non solidified)
10.3 Gel Electrophoresis
5uL dye of 6x loading dye (purple)
65˚ C for 5 min starting time 349 pm -> 354 pm
ice immediately then placed in vortex for 15 seconds at 10,000 rpm
9uL DI H2O 1uL ladder 2uL dye
1000bp
100 bp
Began electrophoresis at 505 pm -> 545pm electrophoresis complete
Photos were taken of the gel on the blue light transilluminator
Gel placed back in electrophoresis for 10 minutes more 610 pm -> 620 pm
more photos taken
Nov 8
Protocol 8.1 Mounting Phage Samples for TEM and Staining with Uranyl Acetate
1 hour centrifuge at 4˚C from 3:46 pm -> 4:46 pm
pulled at 447 pm
supernate extracted, then added 100uL of buffer
4:57 pm placed in freezer 4˚C until 5:45 pm
placed at 10uL lysate on grid at 5:52
5-7 minutes required time to leave on grid due to pfu/mL calculation of 4.0e-7
wicked paper, wash 2 times with H2O
6:00 pm 2 minutes -> 6:02 pm wicked paper -> 6:03 pm -> 6:05 pm wicked paper
6:06 pm 5 uL UA added -> 2 min -> 6:08 pm wicked away access and allowed to dry for 8 minutes.
Grid then placed in A5 slot to await EM
Nov 20
phage data base entry
Previous selected name of Pogo was taken, new name Ryadel
Nov 21
Protocol 7.3 Archiving Your Phage Sample
beads prepared for lysate storage and shipment
DMSO 100 uL mixed with 1.4 mL into a 1.5mL tube
400uL were then extracted and placed in the tube with beads, this was repeated for a total of 3 times, two samples to be sent for analysis and one sample will be kept in the lab freezer for safe keeping
Isolation Protocol 5.1 – Collecting Environmental Samples
-GPS taken via iPhone.
*The original GPS corrdinents were corrected due to inaccuracy.
| Sample | 1. Lake Kirby Abilene TX | 2. Under rotten hay bail Stephenville TX | 3. Home Depot Granbury TX | 4. Base of tree near LDS Stake Center Weatherford TX |
| Collector Name | Travis Miller | Travis Miller | Travis Miller | Travis Miller |
| Date of Collection |
Sep 4, 2017 |
Sep 4, 2017 |
Sep 4, 2017 |
Sep 4, 2017 |
| Sample Type (Soil/Water) | top layer rocky -1/2 down clay-creamy grey color | sand, clay, silt | Peaty | clay, silt |
| Location Description (compost, lake bed, etc) | low amounts of vegetation. High amount of carp feeding | Overgrown old garden, sold underneath rotting hay bails falling apart. | Kellogg Potting Mix Premium Mix for Outdoor Containers | Base of small tree a lot of thick grass covering soil |
| General Location (forest, subdivision, etc) | Shore side of rocky lake Kirby, | Back yard of a remote subdivision | Stacked on a pallet of soil in the garden section of Home Depot, Out side in the direct sun light | near Weatherford High School |
| Specific Location (GPS coordinates) | 32°22’11.7″N 99°43’35.0″W | 32°16’43.6″N 98°08’52.1″W | 32°25’57.0″N 97°46’55.2″W | 32°43’20.3″N 97°48’32.8″W |
| Sample Depth | 1in water 1 in soil | 2 in | 1 in within bag | 2 in |
| Ambient Temperature | 88F | 92F | 93F | 92F
|
Sep 6
Isolation Protocol 5.2 Direct isolation
Protocol was followed, no changes or modifications occurred.
Observations
Direct – Sample #2
8mL of liquid observed
2 layers of soil
top layer dark
bottom layer light
after 10 mixtures x 2000 centrifuge – 3 layers
new layer is light on top
Sep 6
Isolation Protocol 5.3 Plaque Assay
Day 1
protocol followed, only modification was increased time in incubator
sample sat undisturbed for 7 minutes
allowed for a 20 minute for agar solidification
placed in incubator 3pm 37C
Day 2
Direct Isolation pulled after 96 hours incubation and placed in fridge
Sep 6
Isolation Protocol 5.5 Enriched Isolation
Both samples #3 and #4 were tested under Enriched conditions
#3 observations – 35 mL Enriched solution
floating particles
liquid is cloudy and dark brown
two observable soil layers
top layer – near black
bottom layer – dark brown with light brown
After 10 minutes centrifuge x2000 – gray color particle at bottom formed
#3 filtration – 25 mL
observable yellow tint
#4 observations 37.5 mL Enriched Solution
3 layers formed, grass floating in mixture
top layer – gray material with mix of roots and grass
middle layer – layer of light and dark material
bottom layer – combination of light and dark
#4 filtration – 25 mL
faint tint of yellow
Isolation Protocol 5.6 Spot Test
First spot test completed, procedure was followed. Second spot test preformed procedure was modified
Direct Sample placed #1 spot of 9 spot agar plate
Enriched #3 placed on both the 5 and 7 spot
48 hours incubating – non turbid, yellow tint, all collection of light yellow debris/bacteria
Enriched #4 48 hours incubating, turbid, creamy yellow, small yellow debris present
Two plates were spotted.
First plate
1-3 spots were #3
4-6 spots were control
7-9 spots were #4
After 48 hours, 24 in the incubator and 24 in the fridge, both plates shared evidence of phage. Plate 1 which contained only enriched and control showed phage in sections 1-2-4-5-7-8 and 9-6.
Plate #2 was needed due to a spot error, I forgot to add Direct Isolation sample on plate #1 for spot test. Placed in incubator at the same time as Plate #1, it was also taken out and placed in the fridge as well at the same time.. However, prior to incubation and upon cleaning my workstation I bumped the plate and fell into the floor. The lid did come off which may cause a contamination or a shift in the spots made. Dr. Edwards stated not to worry and to carry on with the experiment. Plate #2 was labeled similar to #1 with 9 sections, section 1 and 3 were spotted with Direct Isolation. A control was placed on section 5 and sample #3 at section 7 and sample #4 was placed in section 9. Sections 2,4,6,8 were all left empty to prevent cross contamination.
After 48 hours, 24 in incubator and 24 in fridge phage was present in direct sections 1 and 3. #3 sample showed evidence of phage in sections 4-5-7-8. This may have been due to the drop. Sample #4 showed no sign of phage growth or progress. Splatter dots were also visible throughout the plate, also attributed to the drop.
Potential mess-ups of class and corrections to be made.
a lot of shifted spots
-to correct problem we all change protocol to 30 minute wait time as opposed to 20 minutes, this will allow more time to set and dry as well as soak in
– Dr. Edwards is testing incubator to ensure incubator is plumb
– to help prevent shift we will also only spot 5mL not 10 as directed
Spot Test 2
Plate labeled in 9 sections.
Section 1 – Direct
Section 3 – sample #3
Section 5 – control
Section 7 & 9 – sample #4
Sample #4 was placed in two sections due to previous spot test. I was curious to know if #4 could actually yield a phage since Direct and #3 sample produced phage from the last spot test, I gave #4 sample two spots.
#4 sample did not yield any phage, Direct and #3 samples once again did yield sign of phage.
Sep 20
Purification Protocol 6.1 Plaque Assay for Purification
Purification Protocol 6.2 Serial Dilution (Direct isolation)
Isolation Protocol 5.4 Picking Plaque
Purification Protocol 6.3 Collecting Plate Lysates
– allowed several colleagues to pick #3 smaple, sample shared with Dannielle, Miranda and Musgrave
-Sample #3 was given to Miranda to carry on into the SEA-PHAGE program
plague picked placed in 1.5mL tube with 100mL of buffer
placed 90 uL of buffer in six 1.5 centrifuge tubes
direct isolation sample picked 10 uL placed in 10^-1 vortex 5 seconds
10^-1 extracted 10 uL placed into the 10^-2 vortex continued steps until 10^-6
gathered six 250 uL of bacteria inoculated each tube of bacteria with the centrifuge tube diluted phage
top agar -3 mL into tube with bacteria and phage
plate onto agar allow to sit 20-30 mins start time 1030-1055 am
Sep 22
Purification Protocol 6.2 Serial Dilutions
Observations over Sept 20th procedure pulled plaque assays 24 hours after incubation placed in fridge
small sign of phage present in 10^-1, 10^-2, 10^-3 no sign of phage in 10^-4, 10^-5, 10^-6
Dr. Pierce informed my dilutions looked good but perhaps my phage needed more incubation time because it was so small it was called “slow grower” placed back in incubator 25th of Sept 10 am left until 9 am Sept 27th
results on serial dilutions
10^-1 hard to see visible virus bacteria
10^-2 virus apparent in bacterial carpet extremely webbed will use 10^-2 for webbed plate collectedly lysate protocol 6.3
10^-3 virus still present appears to look like swiss cheese
10^-4 prior to another 2 days of incubation there is no visible virus now 10^-4 is countable
10^-5 no virus present prior to last incubation cycle 10^-5 showed signs of isolated virus to a single plaque
10^-6 no visible sign of phage no more incubation will be tested because web plate and isolated virus have been achieved
Sept 27
Purification Protocol 6.3 Collecting Plate Lysates
all protocol was followed as directed
web plate was used on 10^-2 dilution finished flooding at 945 am
at 3 pm 5.5 mL recovered from flooded plate
plaque assay 3 mL of top agar 250 mL bacteria mixed to create bacterial carpet let sit 20 mins during that time all serial dilution were made all the way from 10^-1 to 10^-8
spots were made with each dilution and a control with a total of 9 dilutions being tested
spots 10^-1, 10^-2, 10^-3, 10^-4 showed visible spots after an incubation time of 26 hours
speaking to Dr. Edwards he instructed me the plate incubated too long and re-do spot checking every 6 hours
Oct 2
Purification Protocol 6.4 Spot Titer
spot titer used previous serial dilutions spotted plate with 10^-1 to 10^-8 as well as the control
allowed plate to sit for 30 mins once spotted to allow for absorption and then placed in incubator at 1040 am will check again within 6 hours also at 430 pm
returned at 430 pm no visible sign of any bacteria or phage will allow to sit over night and check early morning
Oct 3
Purification Protocol 6.5 Full Plate Titer
dilutions 10^-4, 10^-5, 10^-6
10^-5 showed trace amounts of phage present
10^-4, 10^-6 full plate titers will be made along with 10^-5 to better determine what dilution to full plate titer is most sufficient
plates were still slightly cold from fridge in order to have a better plate skin contact was used to warm up the plastic
plaque assay made at 10 am placed in incubator at 1020 am
Oct 4
after another 24 hours at exactly 910 am plates pulled for observation no growth or phage detected in 10^-4, 10^-5, 10^-6
plates returned to incubator at 950 am another round of incubation
Oct 5
all plates pulled at 920 am after another 24 hours of incubation totaling incubation so far for full plate titer 1 day 22 hours 55 mins
plates 10^-4, 10^-5, 10^-6 are now within counting
10^-4 counted 23
10^-5 counted 10
10^-6 counted 2
instructed by Dr. Edward to allow another cycle of incubation
placed in incubator Oct 5th at 1030 am
extracted plates Oct 6th 1 pm
Oct 6
8 mL phage buffer placed on agar for flooding at 140 pm allowed to sit until 420 pm tome which letting sat 2 hours 40 mins
was only able to extract 3.5 mL of 8 mL
loss of buffer could be due to dry agar absorbing liquid for rehydration another possibly for loss would be filter absorption
after another round of incubation 10^-4 dilution yielded 40 plaques this number will be used to calculate a PFU per mL
estimated plaques on the full plate to create webbed plate would be roughly 4000 plaques per agar
40 pfu / (.01 mL) (1e-4) – – 40 pfu / (1e-6 mL) = 40e-6 = 4e-7 pfu/mL
Oct 9
Protocol 7.1 – Making webbed plate from a lysate of known Titer
Estimated 4000 plaques per plate
volume needed mL lysate = 4000/ 4e-7 pfu/mL
1e-4 mL lysate
1e-4 * 1000 = .1 uL
Webbed plates
.1 uL calculated to be ideal amount for proper webbed plate
It is ideal to test 10x more and 10x less than the amount calculated of lysate to determine if calculating are accurate.
In order to produce enough lysate for analysis it is recommended to have 8-10 mL.
Using a total of 6 plates, 2 plates per dilution.
Dilutions being .01 uL, .1uL, and 1uL
Full plate titer dilution of 10^-4
- 10 uL used to create a .1uL and .01uL for webbed plate
- plaque assay, several dilutions and webbed plate
- 6 plates used 2 per concentration
- 9uL added to tubed, 1uL added to 10^-4 to produce a 1uL dilution
- transferred in normal plaque assay step added to 3mL of top agar and plated
- plating completed at 135pm allowed to sit for 20 minutes for solidification before placing in incubator
- started incubation at 2 pm
Oct 10
plates extracted form incubator at 340 pm for a total incubation time of 25 hours 40 minutes. All plates contain phage.
Oct 11
1uL concentration has the most potential to create a webbed plate
plate left out at room temperature from 340pm to 920 am, this allowed for growth but at a slower rate as opposed to the incubator. Total incubation time for plates
1 day 1 hr 40 min in incubator + 17 hrs 40 min = total growth time 1 day 18 hours 20 minutes
Oct 12
Flooded webbed plated of the 1uL sample used collecting lysate protocol to collect total lysate from flooded plated. 14mL collected lysate, placed in fridge
Oct 17
Dr. Edwards and Dr. Pierce pleased with progress, suggested to do another spot test and full plate titer to verify results.
Followed protocol of spot titer and serial dilution protocol
Oct 18
Dilutions 10^-5 and 10^-6 both best based on countability, pulled at 1015 am
Oct 20
Full plates of 10^-5 and 10^-6 used for plate, however, no plates were available at this time.
Oct 23
Full plate titer on 10^-4, 10^-5, 10^-6 plated
retested the serial dilution
plaque assay
10 uL into 250 uL of bacteria incubation started at 1050 am
Oct 24
plates taken out of incubator at 1210 pm for incubation time of 25 hours 20 minutes
phage present on all dilutions, 10^-4 dilution appears to be the best webbed.
Originally 10^-2 dilution was the best webbed plates it appears to have pushed back the dilution to 10^-4
Oct 25
Procedure 9.1 Phage DNA Extraction
procedure followed as directed in Phage Discovery Guid
Nov 6
Qubit ds DNA Analysis kit
2 .5mL tubes
1 master mix 1.5mL tube
Labeled tube 1 and 2 with master
Dilute 1:200 of Quibit buffer
– add 796 uL buffer into tube master mix
– added 4 uL reagent to same tube
-10uL standard in new tube
- added 10 uL standard into tube 2
- add 190 uL master mix to each, mix well
-add 2uL DNA sample in new tube
-add 198uL mix to tube
vortex 4 tubes -> 2 standards
put samples in Quibit reader
Quibit reader calculated DNA to be 99ng/uL
Nov 6
Restriction Enzyme
| .5 g | mL | 1000 uL |
| 99 ug | 1mL |
= 5.05 uL
Began incubation at 1229 pm, 10 minutes at 65˚ C
25uL H2O added to all 6 tubes along with 2.5uL reaction buffer
| 1 | 2 | 3 | 4 | 5 | 6 |
| BamHi | ClaI | EcoRI | Hae III | Hindi III | Sal I |
| red | green | clear | green | blue | red |
| 3.1 | cut smart | EcoRI Buffer | cut smart | 2.1 | 3.1 |
incubate at 37˚ C for 60 minutes
126pm -> 226 pm
Agar Gel
.8% gel
1% = 1g/100mL
.8g/100mL -> .4g/50mL = 400mg/50mL
8% = x needed / 100mL -> .8*50mL/100 = .4g
Buffer 1x dilute TBE
Final 300mL
30mL buffer 5mL buffer
270mL H2O 45 mL H2O
EtBa
(300mL)(.5ug/mL EtBa) = x 10mg/mL
actual ager = .4003g
2uL EtBa into gel (non solidified)
10.3 Gel Electrophoresis
5uL dye of 6x loading dye (purple)
65˚ C for 5 min starting time 349 pm -> 354 pm
ice immediately then placed in vortex for 15 seconds at 10,000 rpm
9uL DI H2O 1uL ladder 2uL dye
1000bp
100 bp
Began electrophoresis at 505 pm -> 545pm electrophoresis complete
Photos were taken of the gel on the blue light transilluminator
Gel placed back in electrophoresis for 10 minutes more 610 pm -> 620 pm
more photos taken
Nov 8
Protocol 8.1 Mounting Phage Samples for TEM and Staining with Uranyl Acetate
1 hour centrifuge at 4˚C from 3:46 pm -> 4:46 pm
pulled at 447 pm
supernate extracted, then added 100uL of buffer
4:57 pm placed in freezer 4˚C until 5:45 pm
placed at 10uL lysate on grid at 5:52
5-7 minutes required time to leave on grid due to pfu/mL calculation of 4.0e-7
wicked paper, wash 2 times with H2O
6:00 pm 2 minutes -> 6:02 pm wicked paper -> 6:03 pm -> 6:05 pm wicked paper
6:06 pm 5 uL UA added -> 2 min -> 6:08 pm wicked away access and allowed to dry for 8 minutes.
Grid then placed in A5 slot to await EM
Nov 20
phage data base entry
Previous selected name of Pogo was taken, new name Ryadel
Nov 21
Protocol 7.3 Archiving Your Phage Sample
beads prepared for lysate storage and shipment
DMSO 100 uL mixed with 1.4 mL into a 1.5mL tube
400uL were then extracted and placed in the tube with beads, this was repeated for a total of 3 times, two samples to be sent for analysis and one sample will be kept in the lab freezer for safe keeping