Difference: ProcedureEvolvabilityMutationAccumulation (3 vs. 4)

Revision 42011-05-19 - LindseyWolf

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META TOPICPARENT name="ProtocolList"

Day 1: Plating the Mixed Population

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Day 2: Picking Colonies

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We'll start off picking 24 colonies. These will end up being our 24 replicates that are passaged simultaneously day-to-day.
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We'll start off picking 12 colonies. These will end up being our 12 replicates that are passaged simultaneously day-to-day.
 
  1. Get out a sterile 384 well freezer plate. Label with strain and date. In the top row, add 50μl 80% glycerol. Sterility is very important here.
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  1. Choose the 24 colonies closest to the written date on the plate that are far enough away from other colonies to be picked without contamination.
  2. Using a sterilized, rounded pasteur pipet, pick each of the 24 colonies and spin it in a glycerol well.
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  1. Choose the 12 colonies closest to the written date on the plate that are far enough away from other colonies to be picked without contamination.
  2. Using a sterilized, rounded pasteur pipet, pick each of the 12 colonies and spin it in a glycerol well. Store plate in the -80C freezer.
 
  1. With a sterile loop touch the remainder of the colony on the plate and streak onto a new MG plate, in a circular fashion, diluting as you go around.
  2. Let grow overnight at 37C.

Day 3: Passaging Colonies

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  1. Pick the last single colony and passage onto a new MG plate.
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  1. For each of the 12 plates: pick the last single colony and passage onto a new MG plate with a sterile toothpick.
  2. Again, streak onto a new MG plate, in a circular fashion, diluting as you go around.
  3. Repeat this passaging each day.
 

Freeze Clones

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  1. Every 4 days, before passaging pull out the freezer plate from the -80C freezer and, in the next row down, add 50μl 80% glycerol to each well.
  2. Using a sterilized, rounded pasteur pipet, pick each of the 12 colonies and spin it in a glycerol well. Return plate to the -80C freezer.

VNTR analysis!

Note to self: Talk to Robin about designing labeled 5' primers, etc!

 
Number Sequence Position Fluorescent Tag?
1 (CA)6 rhaT-rhaD  
2 (CA)12 rhaT-rhaD  
 
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