Lidstrom:EMS Mutagenesis

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(The first time you try it on your organism)
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* It is good to do an exposure time kill curve.   
* It is good to do an exposure time kill curve.   
** For a given amount of cells and EMS, the number of viable cells will decrease with exposure time.
** For a given amount of cells and EMS, the number of viable cells will decrease with exposure time.
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** You can decide how mutated you want your cells to be when you do your selection or phenotype hunting.  This kill curve will help you know what exposure time correlates to what level of mutation.  
+
** You can decide how mutated you want your cells to be when you do your selection or phenotype hunting.  This kill curve will help you know what exposure time correlates to what level of mutation.
 +
** plate 2uL on top of plate and 20uL on bottom.  This is easier than doing dilutions and should give you a good feel for the kill curve given an appropriately dilute suspension of cells.
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*** These culture volumes work well for a SIP3-4 culture grown overnight on methanol from a scraping of a few dozen colonies.
== Sample Protocol ==
== Sample Protocol ==

Revision as of 09:15, 26 June 2014

Back to Protocols

Contents

Overview

  • EMS is a liquid mutagen
    • Work in the fume hood and use special precautions!
  • It tends to produce a certain type of mutations

Tips

  • Use enough cells that you can see the biomass pellet after spinning down the culture to remove the EMS solution.
  • Use clear eppendorf tubes to hold the cells in so you can see the biomass pellet.
    • One could use opaque tubes to reduce EMS's reaction with light, but the protection from light probably isn't worth the convenience of

Health & Safety

  • EMS is a liquid mutagen
    • Work in the fume hood and use special precautions!
  • EMS can be slowly inactivated by mixing with a sodium thiosulfate + NaOH mixture.
  • Discard waste in a specially labeled bottle.
    • Neutralize the pH before turning it into EH&S.
    • Recipes found online vary a bit, but they are about _____:
  • Leave all plastic tips and gloves used for the experiment in the fume hood a day or so after use. Double bag them and discard them in the normal trash stream after that.
    • Some protocols instruct you to soak pipette tips, etc. in a sodium thiosulfate + NaOH mixture for a while before discarding.

The first time you try it on your organism

  • It is good to do an exposure time kill curve.
    • For a given amount of cells and EMS, the number of viable cells will decrease with exposure time.
    • You can decide how mutated you want your cells to be when you do your selection or phenotype hunting. This kill curve will help you know what exposure time correlates to what level of mutation.
    • plate 2uL on top of plate and 20uL on bottom. This is easier than doing dilutions and should give you a good feel for the kill curve given an appropriately dilute suspension of cells.
      • These culture volumes work well for a SIP3-4 culture grown overnight on methanol from a scraping of a few dozen colonies.

Sample Protocol

Open Questions

  • Should you spin down the cells to remove the culture supernatant they were growing in?
    • Yes, if:
      • it has food you don't want them to have access to after mutagenesis
      • you are concerned that EMS could react with compounds in the media
      • you aren't in a rush and want to lean toward a more consistent but time consuming workflow.
    • It is probably fine to skip this step most of the time though.

Misc Notes

  • The fact that EMS is a liquid is nice for health reasons.
    • You don't have to weigh it, so you won't inhale powder or accidentally leave powder behind in the lab or on your clothes
  • ENU is a similar chemical mutagen.
  • UV mutagenesis is safer to perform and should be considered.
    • A similar kill curve can be tested.
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