Difference between revisions of "Kill all but spores"

From PombEvolution
Jump to navigationJump to search
m (I Just added a step in the protocol to make it a bit more clear)
m
Line 4: Line 4:
 
== Glusulase ==
 
== Glusulase ==
 
Glusulase ([http://www.perkinelmer.com NEE154001EA PerkinElmer]) is an enzyme mix that digests cell walls of fungi.
 
Glusulase ([http://www.perkinelmer.com NEE154001EA PerkinElmer]) is an enzyme mix that digests cell walls of fungi.
# Scrape cells of a mating plate ([[EMM-N]]) and suspend these in 0.5 ml sterile water
+
# Scrape cells of a mating plate ([[EMM-lowN]]) and suspend these in 150 μl sterile water
# add 8 ul Glusulase (use the aliquot in the 15ml tube in the door of fridge 1)
+
# add 8 μl Glusulase (use the aliquot in the 15ml tube in the door of fridge 1)
 
# incubate overnight at 32°C
 
# incubate overnight at 32°C
 
# check if the cells are dead
 
# check if the cells are dead
# spin down the cells and resuspend in water
+
# spin down the cells and re-suspend in water
 +
# Plate out in appropriate dilution on YES to ~100-5000 spores/plate
  
 
== 30% ethanol treatment ==
 
== 30% ethanol treatment ==
Line 15: Line 16:
 
# incubate at room temperature for 30 minutes
 
# incubate at room temperature for 30 minutes
 
# spin down the cells at ~1800xg
 
# spin down the cells at ~1800xg
# remove supernatant and resuspend cells in 1 ml sterile water
+
# remove supernatant and re-suspend cells in 1 ml sterile water
 +
# Plate out in appropriate dilution on YES to ~100-5000 spores/plate
  
 
== or do both! ==  
 
== or do both! ==  
 
Even thought Glusulase does a good job, if only sexual offspring is required, on top of the enzyme treatment the ethanol should be performed.
 
Even thought Glusulase does a good job, if only sexual offspring is required, on top of the enzyme treatment the ethanol should be performed.
# after the ON incubation in glusulase, spin down cells
+
# Incubate in 30% ethanol doing:
## If you do this, you don't need to spin down cells. You can calculate the amount of 96% ethanol needed to reach a concentration of 30% in the end.
+
## Add 96% ethanol; you can calculate the amount of 96% ethanol needed to reach a concentration of 30% in the end (~70μl 96% when using 150μl water or 230μl for 0.5ml water)
# resuspend in 30% ethanol
+
## OR after the incubation in Glusulase, spin down cells, remove supernatant and re-suspend in 30% ethanol
 
# continue at step 2 of the ethanol treatment
 
# continue at step 2 of the ethanol treatment
  
 
[[Category:Protocols]]
 
[[Category:Protocols]]

Revision as of 17:14, 20 January 2021

Crosses between cells result in asci, but many of the haploid cells will be maintained and also diploids that did not yet sporulate might be hanging around. To assure only spores are maintained, a double treatment can be performed.


Glusulase

Glusulase (NEE154001EA PerkinElmer) is an enzyme mix that digests cell walls of fungi.

  1. Scrape cells of a mating plate (EMM-lowN) and suspend these in 150 μl sterile water
  2. add 8 μl Glusulase (use the aliquot in the 15ml tube in the door of fridge 1)
  3. incubate overnight at 32°C
  4. check if the cells are dead
  5. spin down the cells and re-suspend in water
  6. Plate out in appropriate dilution on YES to ~100-5000 spores/plate

30% ethanol treatment

In stead of using Glusulase an ethanol treatment can be performed. This is fast and cheap, but will not break the wall of the asci. This is for the lab strain not required, as the spores will be released from the ascus naturally. For other strains, this has not been studied, so be aware of that single spore colonies might not be so single spore.

  1. Scrape cells from a mating plate and resuspend in 30% ethanol.
  2. incubate at room temperature for 30 minutes
  3. spin down the cells at ~1800xg
  4. remove supernatant and re-suspend cells in 1 ml sterile water
  5. Plate out in appropriate dilution on YES to ~100-5000 spores/plate

or do both!

Even thought Glusulase does a good job, if only sexual offspring is required, on top of the enzyme treatment the ethanol should be performed.

  1. Incubate in 30% ethanol doing:
    1. Add 96% ethanol; you can calculate the amount of 96% ethanol needed to reach a concentration of 30% in the end (~70μl 96% when using 150μl water or 230μl for 0.5ml water)
    2. OR after the incubation in Glusulase, spin down cells, remove supernatant and re-suspend in 30% ethanol
  2. continue at step 2 of the ethanol treatment