Day 1: Plating the Mixed Population

  1. Find microsatellite-containing strains in the -80C freezer. They will be labeled with the appropriate microsatellite sequence located in the rhamnose operon (eg. "(CA)12"). See below for the list of microsatellites.
  2. Using careful sterile technique, dilute 1:1000 in saline by adding 10μl frozen stock in 10ml saline.
  3. Dilute this again 1:10 by adding 1ml of the first dilution in 9ml saline.
  4. Plate 100μl on minimal glucose (MG) plates. Label plates with strain and date.
  5. Let grow overnight at 37C.

Day 2: Picking Colonies

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.
  2. 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.
  3. 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.
  4. 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.
  5. Let grow overnight at 37C.

Day 3: Passaging Colonies

  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

  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  
3 (TA)12 rhaT-rhaD  
4 (A)24 rhaT-rhaD  
5 (AGA)8 rhaT-rhaD  
6 (GGA)8 rhaT-rhaD  
7 (G)9 rhaT-rhaD  
8 (G)12 rhaT-rhaD  

-- Main.LindseyWolf - 18 May 2011

 Barrick Lab  >  ProtocolList  >  ProcedureEvolvabilityMutationAccumulation

Topic revision: r5 - 02 Nov 2011 - 16:08:23 - Main.LindseyWolf
 
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