Difference between revisions of "UW-Stout/Nitrogen Starvation FA21"
(→Experimental Procedure) |
|||
| Line 17: | Line 17: | ||
*10-15ul of each yeast strain | *10-15ul of each yeast strain | ||
*Sterile Water | *Sterile Water | ||
| + | |||
| + | '''Control''': Every yeast strain will be put under the same control to see if their growth differs when placed in a plate containing media with a nitrogen source and placed in a media without of a nitrogen source. | ||
'''Media Solution''' | '''Media Solution''' | ||
| − | * | + | *YNB |
| − | * | + | *CSM |
| − | * | + | *His |
| − | * | + | *Leu |
| − | * | + | *Ura |
| − | * | + | *Agar |
| − | * | + | *H2O |
| − | * | + | *Ammonium Source |
| + | |||
====Procedure for Media Formulation==== | ====Procedure for Media Formulation==== | ||
| Line 35: | Line 38: | ||
*Plate the media into the required amount of plates needed and evenly spread. | *Plate the media into the required amount of plates needed and evenly spread. | ||
| − | ====Experimental Procedure | + | ====Experimental Procedure==== |
#Obtain all necessary materials and make sure equipment is functional and ready for use. | #Obtain all necessary materials and make sure equipment is functional and ready for use. | ||
#Obtain 2 media plates for each yeast strain, 1 plate with ammonium and 1 plate without ammonium for each strain. | #Obtain 2 media plates for each yeast strain, 1 plate with ammonium and 1 plate without ammonium for each strain. | ||
| − | # | + | #label all media plates and PCR tubes. |
| − | # | + | #add 10-15ul of each yeast strain being tested into each PCR tube. |
| − | # | + | #using the 96 well plate take your first yeast strain being tested and add 10ul of yeast and 90ul of sterile water into the first well. |
| − | # | + | #mix thoroughly by pipetting the mix in the well up and down several times |
| − | # | + | #then take 10ul from the first well and place it in the second well and add 90ul of sterile water to the second well. |
| − | # | + | #repeat the steps of mixing each well thoroughly and continuously repeat the dilution process in the succeeding wells until till you reach a total of 8 wells filled with your diluted yeast strain. |
| − | # | + | #then using the specialized multi-channel Micro-pipettor, take 5ul from all 8 wells and place it on each of your 2 media plates for that strain. (you can place more than one row on each plate for best results) |
*repeat steps 5 through 9 for each strain being tested. | *repeat steps 5 through 9 for each strain being tested. | ||
| − | # | + | #after your dilutions have been plated, place all the plates in the incubator for at least 48 hours to let the yeast grow. |
| − | # | + | #after removal from incubator, inspect your yeast plates to see any differences between the separate medias for each strain and if certain strains worked differently than others. |
| − | # | + | #record results |
| − | |||
| − | |||
Revision as of 12:47, 14 December 2021
Contents
Nitrogen Starvation Experiment
Introduction
The purpose of this experiment is to test different variations of yeast and how they adapt to the pressure of starving them of nitrogen which is considered necessary for their growth and how the yeast reacts to alterations in media. If any strains of the yeast react better or worse to starvation and if they are able to grow efficiently without a nitrogen source available.
Protocol
Materials/equipment
- 15 plates of media that contains ammonium (nitrogen source)
- 15 plates of media that does not contain ammonium (no nitrogen source)
- 96 well plate
- Micro-pipettors
- Specialized multi-channel micro-pipette
- Micro pipette tips
- 6 PCR tubes containing wild yeast and 5 knockout strains
- 10-15ul of each yeast strain
- Sterile Water
Control: Every yeast strain will be put under the same control to see if their growth differs when placed in a plate containing media with a nitrogen source and placed in a media without of a nitrogen source.
Media Solution
- YNB
- CSM
- His
- Leu
- Ura
- Agar
- H2O
- Ammonium Source
Procedure for Media Formulation
- On two separate containers, two solutions of media with 1/2 L each will be made. One with Ammonium and one without.
- Add required amount of water followed by all required ingredients in descending order from the materials section.
- Place media solutions in Steam Sterilizer Machine for about 40 minutes.
- Plate the media into the required amount of plates needed and evenly spread.
Experimental Procedure
- Obtain all necessary materials and make sure equipment is functional and ready for use.
- Obtain 2 media plates for each yeast strain, 1 plate with ammonium and 1 plate without ammonium for each strain.
- label all media plates and PCR tubes.
- add 10-15ul of each yeast strain being tested into each PCR tube.
- using the 96 well plate take your first yeast strain being tested and add 10ul of yeast and 90ul of sterile water into the first well.
- mix thoroughly by pipetting the mix in the well up and down several times
- then take 10ul from the first well and place it in the second well and add 90ul of sterile water to the second well.
- repeat the steps of mixing each well thoroughly and continuously repeat the dilution process in the succeeding wells until till you reach a total of 8 wells filled with your diluted yeast strain.
- then using the specialized multi-channel Micro-pipettor, take 5ul from all 8 wells and place it on each of your 2 media plates for that strain. (you can place more than one row on each plate for best results)
- repeat steps 5 through 9 for each strain being tested.
- after your dilutions have been plated, place all the plates in the incubator for at least 48 hours to let the yeast grow.
- after removal from incubator, inspect your yeast plates to see any differences between the separate medias for each strain and if certain strains worked differently than others.
- record results
