Difference between revisions of "UW-Stout/Nonionic Detergent SP22"

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Pilot experiment: Testing NLS effect on WT yeast cells growth
 
  
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Introduction: In this experiment, we will be testing the effect of N-Lauroylsarcosine on the speed of growth of wild type yeast cells. Our objective is to find the concentration of NLS needed to affect the yeast cell growth without killing them all.
+
== '''Pilot experiment:''' ==
−
Materials:   
+
NLS effect on WT yeast cells growth
 +
 
 +
1. '''Introduction:''' In this experiment, we will be testing the effect of N-Lauroylsarcosine on the speed of growth of wild type yeast cells. Our objective is to find the concentration of NLS needed to affect the yeast cell growth without killing them all.
 +
 
 +
2. '''Materials:  '''
 
• (NLS) N-Lauroylsarcosine sodium salt >=94%
 
• (NLS) N-Lauroylsarcosine sodium salt >=94%
 
• Corning COSTAR 96-well clear flat-bottom assay plate
 
• Corning COSTAR 96-well clear flat-bottom assay plate
 
• Wild-type yeast in 2x synthetic complete media at an OD600 of 0.1-0.2
 
• Wild-type yeast in 2x synthetic complete media at an OD600 of 0.1-0.2
−
Equipment:
+
 
 +
3. '''Equipment:'''
 
• Molecular Devices SpectraMax Plus 384 Microplate Reader
 
• Molecular Devices SpectraMax Plus 384 Microplate Reader
−
Procedure:  
+
 
 +
4.'''Procedure:'''
 +
 
1. Make a 0.1% solution of NLS by putting 1µl of a 10% solution of NLS in 99 mL of water.
 
1. Make a 0.1% solution of NLS by putting 1µl of a 10% solution of NLS in 99 mL of water.
 
This is going to give you a 100 mL solution with a 0.1 concentration of NLS.
 
This is going to give you a 100 mL solution with a 0.1 concentration of NLS.
 +
 
2. Use the 0.1% solution of NLS to make 7 different concentrations of N-Lauroylsarcosine (0%, 0.001%, 0.002%, 0.005%, 0.01%, 0.02%, 0.05%). To increase solubility, temperature can be increased to 25-30°C.
 
2. Use the 0.1% solution of NLS to make 7 different concentrations of N-Lauroylsarcosine (0%, 0.001%, 0.002%, 0.005%, 0.01%, 0.02%, 0.05%). To increase solubility, temperature can be increased to 25-30°C.
 +
 
3. Set up 8 wells according to following table.
 
3. Set up 8 wells according to following table.
−
Well A (0%) Well B (0.001%) Well C (0.002%) Well D (0.005%) Well E (0.01%) Well F (0.02%) Well G (0.05%) Well H (0.1%)
+
 
−
Yeast culture 50µl 50µl 50µl 50µl 50µl 50µl 50µl 50µl
+
{| border="1" cellspacing="4" cellpadding="2"
−
0.1% solution of NLS X 0.5µl 1µl 2.5µl 5µl 10µl 25µl 50µl
+
|
−
Deionized Water 50µl 49.5µl 49µl 47.5µl 45µl 40µl 25µl X
+
|Well A (0%)
 +
|Well B (0.001%)
 +
|Well C (0.002%)
 +
|Well D (0.005%)
 +
|Well E (0.01%)
 +
|Well F (0.02%)
 +
|Well G (0.05%)
 +
|Well H (0.1%)
 +
|-
 +
|Yeast culture
 +
|50µl
 +
|50µl
 +
|50µl
 +
|50µl
 +
|50µl
 +
|50µl
 +
|50µl
 +
|50µl
 +
|-
 +
|0.1% solution of NLS
 +
|X
 +
|0.5µl
 +
|1.0µl
 +
|2.5µl
 +
|5.0µl
 +
|10µl
 +
|25µl
 +
|50µl
 +
|-
 +
|Deionized water
 +
|50µl
 +
|49.5µl
 +
|49µl
 +
|47.5µl
 +
|45µl
 +
|40µl
 +
|25µl
 +
|X
 +
|}
 +
 
  
 
4. Set up the plate reader as follows:
 
4. Set up the plate reader as follows:
  
 
Temperature: 30°C
 
Temperature: 30°C
 +
 
Mode: Kinetic
 
Mode: Kinetic
 +
 
Wavelength: 600 nm
 
Wavelength: 600 nm
 +
 
Interval: 5 minutes
 
Interval: 5 minutes
 +
 
Total run time: 24 hours
 
Total run time: 24 hours
 +
 
Shake before read: 30 seconds
 
Shake before read: 30 seconds
  
Line 34: Line 86:
 
-Here is the graph obtained after following the procedure above and putting it in the plate reader for 24 hours:
 
-Here is the graph obtained after following the procedure above and putting it in the plate reader for 24 hours:
  
−
[[:Image:Growth rate of a WT yeast cell when mixed with different concentration of NLS.png]]  
+
[[Image:Growth rate of a WT yeast cell when mixed with different concentration of NLS.png|500px]]  
 
   
 
   
  
 
-Under is a graph with a Y axis scale set to logarithmic so we can find the doubling-time of the WT yeast cell in well B,D,E,F when mixed with a 0.001% solution of NLS:
 
-Under is a graph with a Y axis scale set to logarithmic so we can find the doubling-time of the WT yeast cell in well B,D,E,F when mixed with a 0.001% solution of NLS:
  
−
[[:Image:Growth rate of WT yeast cell with 0.001% NLS (Logarithmic).png]]  
+
 
 +
[[Image:Growth rate of WT yeast cell with 0.001% NLS (Logarithmic) .png|500px]]  
 +
 
  
 
'''Doubling time of Well B, D, E, F'''
 
'''Doubling time of Well B, D, E, F'''
Line 50: Line 104:
  
  
−
-Doubling Time for Well A (0% NLS) and Well C (0.002% NLS):
+
'''Doubling Time for Well A (0% NLS) and Well C (0.002% NLS):'''
  
 
-Td (Well A) = 302 minutes
 
-Td (Well A) = 302 minutes
Line 63: Line 117:
  
 
-Important to note: the concentration used need to be divided by 2 because out of the 100µl solution, 50µl is yeast cell so it dilutes the previous dilution. With that being said, we are technically using 0,0005% NLS.
 
-Important to note: the concentration used need to be divided by 2 because out of the 100µl solution, 50µl is yeast cell so it dilutes the previous dilution. With that being said, we are technically using 0,0005% NLS.
 +
 +
 +
 +
== '''Experiment 2:''' ==
 +
NLS effect on KO yeast cells growth
 +
 +
'''1. Introduction:'''
 +
In this experiment, we will be testing the effect of N-Lauroylsarcosine on the speed of growth of different knocked-out yeast cells. Our objective is to find out if there is any knocked-out genes that are related to the growth of yeast cells.
 +
 +
'''2. Materials:  '''
 +
• 0,001% (NLS) N-Lauroylsarcosine sodium salt >=94%
 +
• Corning COSTAR 96-well clear flat-bottom assay plate
 +
• 9 different Knocked-out yeast cells
 +
 +
'''3. Equipment:'''
 +
• Molecular Devices SpectraMax Plus 384 Microplate Reader
 +
 +
'''4. Procedure:'''
 +
 +
1. Make a 0,1% solution of NLS by putting 1µl of a 10% NLS solution in 99µl of DI water.
 +
 +
2. Mix the 0,1% solution with enough water to dilute the concentration to 0.001% while having a 50µl solution. Then, mix it with 9 different KO yeast cell to make nine different 100µl solution.
 +
 +
3. Set up 9 wells according to following table. Do two experiments of this.
 +
 +
{| border="1" cellspacing="4" cellpadding="2"
 +
|
 +
|Well 1 (YDL109C)
 +
|Well 2 (YGL146C)
 +
|Well 3 (YOR111W)
 +
|Well 4 (YHL029C)
 +
|Well 5 (YDR307W)
 +
|Well 6 (YNL058C)
 +
|Well 7 (YCL049C)
 +
|Well 8 (YGR079W)
 +
|Well 9 (YBL113C)
 +
|-
 +
|KO yeast cell
 +
|50µl
 +
|50µl
 +
|50µl
 +
|50µl
 +
|50µl
 +
|50µl
 +
|50µl
 +
|50µl
 +
|50µl
 +
|-
 +
|0.1% solution of NLS
 +
|0.5µl
 +
|0.5µl
 +
|0.5µl
 +
|0.5µl
 +
|0.5µl
 +
|0.5µl
 +
|0.5µl
 +
|0.5µl
 +
|0.5µl
 +
|-
 +
|Deionized Water
 +
|49.5 µl
 +
|49.5 µl
 +
|49.5 µl
 +
|49.5 µl
 +
|49.5 µl
 +
|49.5 µl
 +
|49.5 µl
 +
|49.5 µl
 +
|49.5 µl
 +
|}
 +
 +
4. Set up the plate reader as follows:
 +
 +
Temperature: 30°C
 +
 +
Mode: Kinetic
 +
 +
Wavelength: 600 nm
 +
 +
Interval: 5 minutes
 +
 +
Total run time: 24 hours
 +
 +
Shake before read: 30 seconds
 +
 +
5. Transfer the assay plate to the reader and read for 24 hours.
 +
 +
6. Collect the data and create a graph to compare results.
 +
 +
-Here is the graph for experiment 1 obtained after following the procedure above and putting it in the plate reader for 24 hours:
 +
 +
[[File:Growth rate of different KO yeast cell when mixed with a 0.001% concentration solution of NLS.png|500px]]
 +
 +
-Under is a graph with a Y axis scale set to logarithmic so we can find the doubling-time of the KO yeast cell when mixed with a 0.001% solution of NLS:
 +
 +
[[File:Growth rate of KO yeast cell at 0.001% NLS (Logarithmic).png|500px]]
 +
 +
'''Doubling time of Well 1-9:'''
 +
 +
Td (Well 1):  580 minutes
 +
Td (Well 2):  481 minutes
 +
Td (Well 3):  412 minutes
 +
Td (Well 4):  448.1 minutes
 +
Td (Well 5):  537.8 minutes
 +
Td (Well 6):  570.6 minutes
 +
Td (Well 7):  423 minutes
 +
Td (Well 8):  1766 minutes
 +
Td (Well 9):  1209 minutes

Latest revision as of 12:29, 5 May 2022


Pilot experiment:

NLS effect on WT yeast cells growth

1. Introduction: In this experiment, we will be testing the effect of N-Lauroylsarcosine on the speed of growth of wild type yeast cells. Our objective is to find the concentration of NLS needed to affect the yeast cell growth without killing them all.

2. Materials: • (NLS) N-Lauroylsarcosine sodium salt >=94% • Corning COSTAR 96-well clear flat-bottom assay plate • Wild-type yeast in 2x synthetic complete media at an OD600 of 0.1-0.2

3. Equipment: • Molecular Devices SpectraMax Plus 384 Microplate Reader

4.Procedure:

1. Make a 0.1% solution of NLS by putting 1µl of a 10% solution of NLS in 99 mL of water. This is going to give you a 100 mL solution with a 0.1 concentration of NLS.

2. Use the 0.1% solution of NLS to make 7 different concentrations of N-Lauroylsarcosine (0%, 0.001%, 0.002%, 0.005%, 0.01%, 0.02%, 0.05%). To increase solubility, temperature can be increased to 25-30°C.

3. Set up 8 wells according to following table.

Well A (0%) Well B (0.001%) Well C (0.002%) Well D (0.005%) Well E (0.01%) Well F (0.02%) Well G (0.05%) Well H (0.1%)
Yeast culture 50µl 50µl 50µl 50µl 50µl 50µl 50µl 50µl
0.1% solution of NLS X 0.5µl 1.0µl 2.5µl 5.0µl 10µl 25µl 50µl
Deionized water 50µl 49.5µl 49µl 47.5µl 45µl 40µl 25µl X


4. Set up the plate reader as follows:

Temperature: 30°C

Mode: Kinetic

Wavelength: 600 nm

Interval: 5 minutes

Total run time: 24 hours

Shake before read: 30 seconds

5. Transfer the assay plate to the reader and read for 24 hours. Notes • Results:

-Here is the graph obtained after following the procedure above and putting it in the plate reader for 24 hours:

Growth rate of a WT yeast cell when mixed with different concentration of NLS.png


-Under is a graph with a Y axis scale set to logarithmic so we can find the doubling-time of the WT yeast cell in well B,D,E,F when mixed with a 0.001% solution of NLS:


Growth rate of WT yeast cell with 0.001% NLS (Logarithmic) .png


Doubling time of Well B, D, E, F

-Td (Well B)= 547 minutes -Td (Well D) = 1442 minutes -Td (Well E) = 1086 minutes -Td (Well F) = 496 minutes -Td (average) = 893 minutes


Doubling Time for Well A (0% NLS) and Well C (0.002% NLS):

-Td (Well A) = 302 minutes -Td (Well C) = 10 570 minutes


• Analysis and Conclusions:

-According to the graph above and the doubling time found, we noticed that in well A (with only water), the growth of the WT yeast cell was significant with a doubling time of 302 minutes. In Well C (0,002% NLS), practically no growth occurred with having a doubling time of 10 570 minutes. Following this information, we concluded that 0,002% NLS was too concentrated for any growth to occur.

-Furthermore, after seeing that well B’s growth rate graph and its doubling time (893 minutes) were situated in between A’s and C’s, we concluded that 0,001% was the concentration we were going to use.

-Important to note: the concentration used need to be divided by 2 because out of the 100µl solution, 50µl is yeast cell so it dilutes the previous dilution. With that being said, we are technically using 0,0005% NLS.


Experiment 2:

NLS effect on KO yeast cells growth

1. Introduction: In this experiment, we will be testing the effect of N-Lauroylsarcosine on the speed of growth of different knocked-out yeast cells. Our objective is to find out if there is any knocked-out genes that are related to the growth of yeast cells.

2. Materials: • 0,001% (NLS) N-Lauroylsarcosine sodium salt >=94% • Corning COSTAR 96-well clear flat-bottom assay plate • 9 different Knocked-out yeast cells

3. Equipment: • Molecular Devices SpectraMax Plus 384 Microplate Reader

4. Procedure:

1. Make a 0,1% solution of NLS by putting 1µl of a 10% NLS solution in 99µl of DI water.

2. Mix the 0,1% solution with enough water to dilute the concentration to 0.001% while having a 50µl solution. Then, mix it with 9 different KO yeast cell to make nine different 100µl solution.

3. Set up 9 wells according to following table. Do two experiments of this.

Well 1 (YDL109C) Well 2 (YGL146C) Well 3 (YOR111W) Well 4 (YHL029C) Well 5 (YDR307W) Well 6 (YNL058C) Well 7 (YCL049C) Well 8 (YGR079W) Well 9 (YBL113C)
KO yeast cell 50µl 50µl 50µl 50µl 50µl 50µl 50µl 50µl 50µl
0.1% solution of NLS 0.5µl 0.5µl 0.5µl 0.5µl 0.5µl 0.5µl 0.5µl 0.5µl 0.5µl
Deionized Water 49.5 µl 49.5 µl 49.5 µl 49.5 µl 49.5 µl 49.5 µl 49.5 µl 49.5 µl 49.5 µl

4. Set up the plate reader as follows:

Temperature: 30°C

Mode: Kinetic

Wavelength: 600 nm

Interval: 5 minutes

Total run time: 24 hours

Shake before read: 30 seconds

5. Transfer the assay plate to the reader and read for 24 hours.

6. Collect the data and create a graph to compare results.

-Here is the graph for experiment 1 obtained after following the procedure above and putting it in the plate reader for 24 hours:

Growth rate of different KO yeast cell when mixed with a 0.001% concentration solution of NLS.png

-Under is a graph with a Y axis scale set to logarithmic so we can find the doubling-time of the KO yeast cell when mixed with a 0.001% solution of NLS:

Growth rate of KO yeast cell at 0.001% NLS (Logarithmic).png

Doubling time of Well 1-9:

Td (Well 1): 580 minutes Td (Well 2): 481 minutes Td (Well 3): 412 minutes Td (Well 4): 448.1 minutes Td (Well 5): 537.8 minutes Td (Well 6): 570.6 minutes Td (Well 7): 423 minutes Td (Well 8): 1766 minutes Td (Well 9): 1209 minutes

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