Difference: ProcedureEvolvabilityMutationAccumulation (1 vs. 5)

Revision 52011-11-02 - LindseyWolf

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META TOPICPARENT name="ProtocolList"

Day 1: Plating the Mixed Population

Revision 42011-05-19 - LindseyWolf

Line: 1 to 1
 
META TOPICPARENT name="ProtocolList"

Day 1: Plating the Mixed Population

Line: 10 to 10
 

Day 2: Picking Colonies

Changed:
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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.
>
>
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.
Changed:
<
<
  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.
>
>
  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

Changed:
<
<
  1. Pick the last single colony and passage onto a new MG plate.
>
>
  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

Added:
>
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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  

Revision 32011-05-19 - LindseyWolf

Line: 1 to 1
 
META TOPICPARENT name="ProtocolList"

Day 1: Plating the Mixed Population

Line: 6 to 6
 
  1. Using careful sterile technique, dilute 1:1000 in saline by adding 10μl frozen stock in 10ml saline.
  2. Dilute this again 1:10 by adding 1ml of the first dilution in 9ml saline.
  3. Plate 100μl on minimal glucose (MG) plates. Label plates with strain and date.
Changed:
<
<
  1. Let grow overnight.
>
>
  1. Let grow overnight at 37C.
 

Day 2: Picking Colonies

Line: 14 to 14
 
  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 24 colonies closest to the written date on the plate that are far enough away from other colonies to be picked without contamination.
Changed:
<
<

Freeze "Ancestral" Clones

>
>
  1. Using a sterilized, rounded pasteur pipet, pick each of the 24 colonies and spin it in a glycerol well.
  2. 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.
  3. Let grow overnight at 37C.
 

Day 3: Passaging Colonies

Added:
>
>
  1. Pick the last single colony and passage onto a new MG plate.
 

Freeze Clones

Number Sequence Position Fluorescent Tag?

Revision 22011-05-18 - LindseyWolf

Line: 1 to 1
 
META TOPICPARENT name="ProtocolList"

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.
Added:
>
>
  1. Using careful sterile technique, dilute 1:1000 in saline by adding 10μl frozen stock in 10ml saline.
  2. Dilute this again 1:10 by adding 1ml of the first dilution in 9ml saline.
  3. Plate 100μl on minimal glucose (MG) plates. Label plates with strain and date.
  4. Let grow overnight.
 

Day 2: Picking Colonies

Added:
>
>
We'll start off picking 24 colonies. These will end up being our 24 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 24 colonies closest to the written date on the plate that are far enough away from other colonies to be picked without contamination.

Freeze "Ancestral" Clones

 

Day 3: Passaging Colonies

Added:
>
>

Freeze Clones

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

Revision 12011-05-18 - LindseyWolf

Line: 1 to 1
Added:
>
>
META TOPICPARENT name="ProtocolList"

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.

Day 2: Picking Colonies

Day 3: Passaging Colonies

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

 
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