Difference between revisions of "UW-Stout/pH"

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Intro:
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'''Intro:'''
  
 
This page is dedicated to the test protocol used to view stress on yeast cells in a lowered pH environment.
 
This page is dedicated to the test protocol used to view stress on yeast cells in a lowered pH environment.
 
We started by mixing up 1 molar HCL into distilled water using a pH probe to determine when we reached the desired level. We had pH from 7 down to 4 going in roughly 1 pH step per solution. We the used these in a calibration test where we tested a unmodified wild type BY4735. This gave us a data sheet showing the growth times and effects of the varied pH levels.
 
We started by mixing up 1 molar HCL into distilled water using a pH probe to determine when we reached the desired level. We had pH from 7 down to 4 going in roughly 1 pH step per solution. We the used these in a calibration test where we tested a unmodified wild type BY4735. This gave us a data sheet showing the growth times and effects of the varied pH levels.
  
 
+
----
 
+
'''Materials:'''
Calibration Materials:
 
  
 
Hydrochloric Acid
 
Hydrochloric Acid
  
Yeast Cells To test
+
Yeast Cells To test  
  
 
8 Test Tubes
 
8 Test Tubes
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Ph probe
 
Ph probe
  
Calibration Protocol:
+
Syringe and sterilizing filter for purification.
  
Steps:
 
1) Gather the test tubes and start to label each one with the pH levels accordingly by increments of 1. And label each tube with the first one at 4 and then 5 and then 6 and then for the control and labels it’s neutral base of 7 and have that be the control of the experiment.
 
  
2) Gather the professor and ask him to hover/ help you with the dilution of the sulfuric acid at the according pH levels marked on the test tubes and begin to dilute the acid to the first pH of 4  and then keep diluting the acid by increments of 1 diluted pH to the according tube of labeled up to the ph of 6.
+
----
  
3) Now Either invert the tubes to thoroughly mix the solution or put it on the vortex to make sure it is mixed.
+
'''Protocol:'''
  
4) Grab the plate and start to pipet out 90 uL of the yeast cell into 12 wells of the plate you are using. And then label Three wells accordingly with the levels of the ph 4 and then  5 and then 6 and then for the base/neutral concentration of 7 just put the cell into the wells without any acid. And then pipet out 10 ul of the first acid concentration of 4 into the labeled well of ph 4 and then same on for the other labeled levels of ph.
+
1) Gather Test tubes, DI water, 1 Molar HCL, and pH probe.
  
5) After it has sit for about a day look at each well and observe the level of growth compared to the control and write down in your notebook on what you see and the level of damage the different levels of ph caused.
+
2) Using the pH prob, slowly dispense HCL into a beaker of DI water while stirring the solution until the desired pH has been reached. (For this lab we used pH levels of roughly 4,5,6,7)
  
 +
3) Then using these solutions, mix with your wild type cells, in a test plate. We did three wells per pH. This data is going to be your calibration to tell which pH has the most effect on the wild type.
 +
 +
For this lab as you can see in the data table the only pH wich really killed off the cells was in well 12, which contained pH of 4. After seeing this data, we decided to use the pH 4 solution for our strain test.
 +
Here is the calibration data:
  
  
Raw Calibration Data:
+
'''Raw Calibration Data:'''
  
 
[[File:Raw Data.PNG]]
 
[[File:Raw Data.PNG]]
  
  
 +
Protocol for strain test.
 +
 +
1) Using a sterilizing filter on a syringe, sterilize a few test tubes of solution (pH 4). Just so that you can be sure that the only growth in your plate is from wanted cells.
 +
 +
2)Take your sterilized water, obtain a test plate and fill 9 wells with solution (anywhere from 50 to 80 micro liters).
  
 +
3)Then in each of the wells deposit a small amount of each cell type that you are testing, (roughly 50 to 30 micro liters, so that the well is at 100 micro liters)
  
Final data:
+
4) in this lab we ussed, one wild type for the control, and then 8 different knock out strains for data
  
  
[[File:Capture.PNG]]
+
'''Final data:'''
  
  
 +
[[File:Final Data.png]]
  
start of data?
 
 
When looking at this mess of a graph we must find the useful bits, which to us  
 
When looking at this mess of a graph we must find the useful bits, which to us  
 
is going to be the doubling times for each line.
 
is going to be the doubling times for each line.
 
In order to find doubling time we must use a doubling time equation which is as follows:
 
In order to find doubling time we must use a doubling time equation which is as follows:
Td=(Tf-Ti)*(((log(2))/(log((q2)/(q1)))
+
Td=(Tf-Ti)*(((log(2))/(log((q2)/(q1))). Below is a table of all of the strands doubling time and strand name in relation to their graph labels.
I will be labeling the data as follows
+
For in depth look at each gene and the reaction to individual data compared to the wild type, check the individual gene pages linked below.
 +
 
 
----                       
 
----                       
                      Time          q value
+
 
Your Favorite Gene:
 
                    1: Ex)              Ex)
 
                    2: Ex)              Ex)
 
 
{| class="wikitable"
 
{| class="wikitable"
 
|-
 
|-
! Gene
+
!Gene
! Time
+
! Doubling Time (in Min)
! q value
+
|-
 +
| F1:BY4735
 +
| Td:560
 +
|-
 +
| F2:YEL035L
 +
| Td:483.73
 +
|-
 +
| F3:YEL002
 +
| Td:267.824
 +
|-
 +
| F4:YFL064C
 +
| Td:3718.50
 +
|-
 +
| F5:YG1235W
 +
| Td:388.57
 +
|-
 +
| F6:YCR100C
 +
| Td:386.59
 
|-
 
|-
| By4735
+
| F7:YFL043W
|  T1:615
+
| Td:511.077
| q1:0.2983
 
 
|-
 
|-
|  
+
| F8:YPL068
| T2:1305
+
| Td:390.84
| q2:0.2983
+
|-
 +
| F9:YMR090W
 +
| Td:251.55
 
|}
 
|}
 +
 +
----
 +
'''Links to individual gene pages'''
 +
 +
YCL002C:[[https://wiki.yeastgenome.org/index.php/YCL002C]]
 +
 +
YCR100C:[[https://wiki.yeastgenome.org/index.php/YCR100C]]
 +
 +
YEL035C:[[https://wiki.yeastgenome.org/index.php/YEL035C]]
 +
 +
YFL064C:[[https://wiki.yeastgenome.org/index.php/YFL064C]]
 +
 +
YGL235W:[[https://wiki.yeastgenome.org/index.php/YGL235W]]
 +
 +
YJL043W:[[https://wiki.yeastgenome.org/index.php/YJL043W]]
 +
 +
YMR090W:[[https://wiki.yeastgenome.org/index.php/YMR090W]]
 +
 +
YPL068C:[[https://wiki.yeastgenome.org/index.php/YPL068C]]

Latest revision as of 11:05, 9 May 2019

Intro:

This page is dedicated to the test protocol used to view stress on yeast cells in a lowered pH environment. We started by mixing up 1 molar HCL into distilled water using a pH probe to determine when we reached the desired level. We had pH from 7 down to 4 going in roughly 1 pH step per solution. We the used these in a calibration test where we tested a unmodified wild type BY4735. This gave us a data sheet showing the growth times and effects of the varied pH levels.


Materials:

Hydrochloric Acid

Yeast Cells To test

8 Test Tubes

Proper PPE

10 ml of 1 molar ACL

Distilled water

Pipetting equipment

Ph probe

Syringe and sterilizing filter for purification.



Protocol:

1) Gather Test tubes, DI water, 1 Molar HCL, and pH probe.

2) Using the pH prob, slowly dispense HCL into a beaker of DI water while stirring the solution until the desired pH has been reached. (For this lab we used pH levels of roughly 4,5,6,7)

3) Then using these solutions, mix with your wild type cells, in a test plate. We did three wells per pH. This data is going to be your calibration to tell which pH has the most effect on the wild type.

For this lab as you can see in the data table the only pH wich really killed off the cells was in well 12, which contained pH of 4. After seeing this data, we decided to use the pH 4 solution for our strain test. Here is the calibration data:


Raw Calibration Data:

Raw Data.PNG


Protocol for strain test.

1) Using a sterilizing filter on a syringe, sterilize a few test tubes of solution (pH 4). Just so that you can be sure that the only growth in your plate is from wanted cells.

2)Take your sterilized water, obtain a test plate and fill 9 wells with solution (anywhere from 50 to 80 micro liters).

3)Then in each of the wells deposit a small amount of each cell type that you are testing, (roughly 50 to 30 micro liters, so that the well is at 100 micro liters)

4) in this lab we ussed, one wild type for the control, and then 8 different knock out strains for data.


Final data:


Final Data.png

When looking at this mess of a graph we must find the useful bits, which to us is going to be the doubling times for each line. In order to find doubling time we must use a doubling time equation which is as follows: Td=(Tf-Ti)*(((log(2))/(log((q2)/(q1))). Below is a table of all of the strands doubling time and strand name in relation to their graph labels. For in depth look at each gene and the reaction to individual data compared to the wild type, check the individual gene pages linked below.


Gene Doubling Time (in Min)
F1:BY4735 Td:560
F2:YEL035L Td:483.73
F3:YEL002 Td:267.824
F4:YFL064C Td:3718.50
F5:YG1235W Td:388.57
F6:YCR100C Td:386.59
F7:YFL043W Td:511.077
F8:YPL068 Td:390.84
F9:YMR090W Td:251.55

Links to individual gene pages

YCL002C:[[1]]

YCR100C:[[2]]

YEL035C:[[3]]

YFL064C:[[4]]

YGL235W:[[5]]

YJL043W:[[6]]

YMR090W:[[7]]

YPL068C:[[8]]